Lipase Catalyzed Aminolysis as

An Entry to Consecutive Multicomponent Reactions

Inaugural-Dissertation

for the attainment of the title of doctor

in the Faculty of Mathematics and Natural Sciences

at the Heinrich Heine University Düsseldorf

presented by

Sidra Hassan

from Lahore, Pakistan

Düsseldorf, September 2014

From the Institute of Organic Chemistry and Macromolecular Chemistry, Heinrich-Heine University, Düsseldorf. Printed with the permission of the Faculty of Mathematics and Natural Sciences of the Heinrich Heine University, Düsseldorf. Research Supervisor: Prof. Dr. Thomas J. J. Müller Co-examiner: Prof. Dr. Jörg Pietruzska Date of Examination:

I hereby declare that the work presented here is reflection of my own independent efforts

and had been conducted without any unauthorized assistance. Wherever contributions and

consultation of other sources are involved, every effort is made to indicate this clearly with

due reference to the literature and acknowledgement of collaborative research and

discussions, if any. Moreover the dissertation has never been submitted in any form to any

other institution.

Düsseldorf, 22.09.2014

(Sidra Hassan)

The present work was conducted during the time period from April 2011 to January 2014, at

the Institute of Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University

Düsseldorf, under the supervision of Prof. Dr. Thomas J. J. Müller.

Part of this work has already been published or submitted for publication or presented as

posters at scientific meetings:

Publication in Scientific Journal

"Three-component Chemoenzymatic Synthesis of Amide Ligated 1,2,3-Triazoles"

S. Hassan, R. Tschersich, T. J. J. Müller, Tetrahedron Lett. 2013, 54, 4641–4644.

Poster Presentations "One-Pot Chemoenzymatic Synthesis of 1,2,3-Triazoles"

Third Annual CLIB-GC Retreat 2012, 22-24.02.2012, Bergisch Gladbach, Germany.

"One-Pot Chemoenzymatic Synthesis of 1,2,3-Triazoles"

Biotrends: Sustainable Industrial Biocatalysis International Congress, 29-30.11.2012,

Dortmund, Germany.

"One-Pot Chemoenzymatic Synthesis of 1,2,3-Triazoles"

Fourth Annual CLIB-GC Retreat, 13-15.02.2013, Lünen, Germany.

"Three-component Chemoenzymatic Synthesis of 1,2,3-Triazoles"

Heidelberg Forum of Molecular Catalysis: International Symposium, 28.06.2013, University

of Heidelberg, Germany.

Oral Presentations

"Development of Chemoenzymatic Amidation-Coupling-Cycloisomerization (ACCI)

Sequence in a One-pot Fashion for the Synthesis of Oxazoles"

First Internal(Düsseldorf/Jülich) CLIB-GC Retreat Mülheim, Germany, 30-31.08.2011.

"One-Pot Chemoenzymatic Synthesis of 1,2,3-Triazoles"

Second Internal (Düsseldorf/Jülich) CLIB-GC Retreat Mülheim, Germany, 23-24.08.2012.

"Application of Chemoenzymatic Catalysis in Multicomponent Reactions"

Ecochem Congress: The Sustainable Chemistry and Engineering Event, Basel Switzerland,

19-21.11.2013.

My Thanks to....

I would like to thank ALMIGHTY for blessing me with the wonderful opportunities in this life

and conferring upon me the confidence and courage to pursue my dreams.

My heartiest gratitude to my research supervisor Prof. Dr. Thomas J. J. Müller, right from the

selection process till now, for his kindness, cooperation, guidance, and for giving me the

opportunity to work in a highly competitive and productive environment on such an

interesting research topic in his work group.

I would like to thank my second supervisor Prof. Dr. Jörg Pietruszka for his approachability

during the research project.

I would like to thank Cluster of Industrial Biotechnology-Graduate Cluster (CLIB-GC) for

providing the financial support for the research project.

I feel deeply obliged to pay my thanks to CLIB-GC coordinator, Dr. Sonja Meyer zu

Berstenhorst, for her continual help especially during my early days in Germany.

I would like to thank Dr. Bernhard Mayer for his benevolence and for being consistently

considerate. My deepest appreciation to all the past and the present members of the work

group for welcoming me so warmly in the group and also for their unconditional support and

help both on personal and professional levels. I would especially like to thank my lab fellows,

Alissa Götzinger and Elena Dirksen, with whom I had a great time both on and off lab

ventures together. My special thanks to all the members of the technical staff for their

technical assistance throughout my research tenure.

This also gives me an opportunity to thank my chemistry teacher and mentor Dr. Fehmida

Tasneem Baqai, who I consider my "professional mother", for always instigating me to

pursue my carrier actively.

Last but not least, I must acknowledge my loving and caring family members back home,

who not only supported me, prayed for my success, but also exhibited great patience and

goodwill throughout the fairly long period of my studies here. Whatever little I have achieved

in my life or strive for achieving, I owe this to my ever supporting mother and brother for their

encouragement and providing me with a constant sense of reliance.

To my mother

for making me certain that it's absolutely normal not to be a normality

...."your longing for Me is My message to you

all your attempts to reach Me

are in reality My attempts to reach you"....

(An excerpt from Chanting the Name by Rumi)

"So it was that I began to study not for praise or good opinion of others, but for the

independence I thought it could give me. My studies were soon my life. They gave me

refuge as well as occupation, for when I was sunk in my books I felt myself not alone, but in

company: a company of enlightened souls whose passion was the enlightenment of others. I

felt privileged to eavesdrop on the discourse of these great minds, and sometimes resented

the intrusion of daily life. I can admit this to you because I know you will understand the

temptation of a scholarly existence. Sometimes I think if I had not taken that road, a tumult of

emotions might have overwhelmed me. Anger, sorrow and loneliness lay in wait on every

side. I had only to stray a step or two from the path and I would be lost to them, and to

despair. For what use are the tears of a few gentle souls against the cruelty of so many?"

(An excerpt from The Einstein Girl by Philip Sington)

Contents

Contents

List of Abbreviations ............................................................................................... 1

1. Summary .............................................................................................................. 5

2. Introduction .......................................................................................................... 9

2.1. Enzyme Catalysis in Organic Solvents ....................................................... 9

2.2. Enhancement of Enzyme Activity via Excipients ........................................ 10

2.3. Stabilization of Enzyme via Immobilization ................................................. 11

2.4. Enzyme Selectivity ..................................................................................... 11

2.5. Chemoenzymatic Multicomponent Reactions (MCRs) ................................ 12

2.5.1. MCRs Utilizing Enzymatic Resolution of Racemic Mixtures ................ 13

2.5.1.1. Ugi Four-Component Reaction .................................................... 13

2.5.1.2. Passerini Multicomponent Reaction ............................................ 16

2.5.1.3. Biginelli Multicomponent Reactions ............................................. 18

2.5.1.4. Synthesis of Cyclic Systems ....................................................... 18

2.5.1.5. Strecker Reaction........................................................................ 20

2.5.2. Enzyme Catalyzed Multicomponent Reactions ................................... 20

3. Aim of the Project ................................................................................................ 25

4. Literature Review ................................................................................................. 27

4.1. Mechanistic Aspects of Lipase Catalyzed Aminolysis ................................. 27

4.2. Development of Lipase Catalyzed Aminolysis ............................................ 28

4.3. Development of Synergy of Biocatalysis with Click Multicomponent

Syntheses of Triazoles ...................................................................................... 37

4.4. Synergy of Biocatalysis with Palladium Catalyzed Coupling Reactions ...... 41

4.5. Heterocyclization of Propargylamides ........................................................ 44

4.5.1. Oxazole Syntheses .......................................................................... 44

4.5.2. Intramolecular Cycloisomerization of Terminal Propargylamides ...... 46

4.5.3. Intramolecular Cycloisomerization of Non-terminal

Propargylamides ....................................................................................... 48

Contents

4.5.4. Coupling and Cycloaddition Reactions of Propargylamides .............. 48

5. Results and Discussion ....................................................................................... 51

5.1. Optimization of Enzyme Catalyzed Aminolysis ..................................... 51

5.1.1. Solvent Selection ............................................................................. 51

5.1.2. Screening of Enzymes ..................................................................... 52

5.1.3. Substrate (Acyl Donor) Selection ...................................................... 54

5.1.3.1. Substrates with Heteroatom Side Chain Linkers ................... 55

5.1.3.2. N-Protected Amino Acids as Ester Substrates ...................... 58

5.1.3.3. Ester Substrates with Heterocyclic Systems ......................... 60

5.1.3.4. Methyl propiolate as Acyl Donor ........................................... 61

5.1.3.5. Long Chain Fatty Acids ........................................................ 62

5.2. Optimized CAL-B (Novozyme® 435) Aminolysis .................................... 63

5.3. Optimization Studies for Chemoenzymatic One-pot Synthesis of

1,4-Disubstituted 1,2,3-Triazoles 5 ................................................................ 64

5.4. Synthesis of Triazoles 5 via In situ Azide Generation ........................... 69

5.5. Optimized Chemoenzymatic One-pot Synthesis of Amide Ligated

1,4-Disubstituted 1,2,3-Triazoles 5................................................................. 70

5.6. Intended Modification of ACCI Sequence............................................... 73

5.6.1. Optimization Studies of Sonogashira Coupling Reaction .................. 73

5.6.2. Cycloisomerization of Propargylamides 3 ......................................... 77

5.6.2.1. Lewis/Protic Acid Mediated Cycloisomerization .................... 77

5.6.2.2. Oxidative Cycloisomerization ............................................... 78

5.7. Optimized Chemoenzymatic One-pot Amidation-Sonogashira

Coupling Sequence ........................................................................................ 79

6. Outlook ................................................................................................................. 84

7. Experimental ........................................................................................................ 86

7.1. General Considerations ........................................................................... 86

7.2. Syntheses of Starting Materials .............................................................. 87

Contents

7.3. General Procedure for CAL-B (Novozyme® 435) Catalyzed

Aminolysis ....................................................................................................... 94

7.4. Analytical Data of Propargylamides 3 .................................................... 95

7.5. Procedure for Chemoenzymatic One-pot Synthesis of Amide Ligated

1,4-Disubstituted 1,2,3-Triazoles 5................................................................. 108

7.6. Analytical Data of Amide Ligated 1,4-Disubstituted 1,2,3-Triazoles 5 .. 110

7.7. General Procedure for Chemoenzymatic One-pot Amidation-

Coupling Sequence ........................................................................................ 130

7.8. Analytical Data of Arylated Propargylamides 6 ..................................... 131

7.9. General Procedure for Chemoenzymatic One-pot Triazole Ligation

to Arylated Propargylamides ......................................................................... 142

7.10. Analytical Data of Triazole Ligated Systems 8 ..................................... 143

8. Molecule Content ................................................................................................. 145

9. References............................................................................................................ 151

List of Abbreviations

1

List of Abbreviations

α alpha

Å Ångström

Ac acyl

ACCI amidation-coupling-cycloisomerization

ADH alcohol dehydrogenase

ANAD sequence anhydrides, acetamide and dienophile sequence

Ar aryl

ATRP atom transfer radical polymerization

BCL Burkholderia cepacia lipase

[BMIm(BF4)] 1-butyl-3-methylimidazolium tetrafluoroborate

[BMIm(PF6)] 1-butyl-3-methylimidazolium hexafluorophosphate

Bn benzyl

Boc t-butoxycarbonyl

br broad

BSA bovine serum albumin

BQ benzoquinone

Bu butyl

°C Degree Centrigrade

CAL Candida antarctica lipase

CAL-A Candida antarctica lipase A

CAL-B Candida antarctica lipase B

CCL Candida cylindracea lipase

CLEA cross-linked enzyme aggregate

CMP cytidine monophosphate

3CR 3 component reaction

4CR 4 component reaction

CRL Candida rugosa lipase

CTP cytidine-5'-phosphate

CuAAC Cu-catalyzed azide-alkyne cycloaddition

Cy cyclohexyl

d doublet

DABCO 1,4-diazabicyclo[2.2.2]octane

dba dibenzylideneacetone

DBU 1,8-diazabicyclo[5.4.0]undec-7-ene

DCE dichloroethane

List of Abbreviations

2

DCM dichloromethane

dd doublet of doublets

DEPT distortionless enhancement by polarization transfer

DFT density functional theory

DHPMs dihydropyrimidones

DIPE diisopropylether

DIPEA diisopropylethylamine

DKPs 2,5-diketopiperazines

DMF N,N-dimethyl formamide

DMSO dimethylsulfoxide

ee enantiomeric excess

EI electron impact

Enz enzyme

eq equivalent

Et ethyl

EWG electron withdrawing group

Gal(NAc) N-acetylglactosamine

GC-MS Gas Chromatography-Mass Spectrometry

Glu glutamic acid

HheC halohydrin dehalogenase

h hour

Hz Hertz

IPr·HCl 1,3-bis(2,6-diisopropylphenyl)imidazolium chloride

IR infrared

J coupling constant

m multiplet

M molar

MALDI matrix assisted laser desorption ionization

MAO-N monoamine oxidase N

MCP multicomponent polymerization

MCRs multicomponent reactions

Me methyl

min Minutes

MML Mucor miehei lipase

mol. sieves molecular sieves

MTBE methyl-t-butyl ether

List of Abbreviations

3

m/z mass to charge ratio

N Normal (solution)

NMR nuclear magnetic resonance

o ortho

p para

P1 P1 protease

PCL Pseudomonas cepacia lipase

PEG polyethylene glycol

PestE thermophilic esterase

PGA penicillin G amidase

PG protecting group

Ph phenyl

PIDA (diacetoxyiodo)benzene

PIFA (bis(trifluoroacetoxy)iodo)benzene

PLE pig liver esterase

PPi pyrophosphate

PPL porcine pancreatic lipase

ppm parts per million

Pr propyl

PTSA p-toluenesulfonic acid

q quartet

quat quaternary

® registered sign

RAL Rhizopus arrhizus lipase

RT room temperature

s singlet

T temperature

t triplet

t tertiary

TBAC tetrabutylammonium chloride

TBDMS t-butyldimethylsilyl

TEMPO 2,2,6,6-tetramethylpiperidine 1-oxide

TFA trifluoroacetyl

Tf triflate

THF tetrahydrofuran

TMEDA N,N,N,N-tetramethylethylenediamine

List of Abbreviations

4

TMG 1,1,3,3-tetramethyl guanidine

TMS trimethylsilyl

v/v volume to volume ratio

w/w weight to weight ratio

XANTPHOS 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

Summary

5

1. Summary

Enzyme catalysis earns its importance owing to its regio-, chemo- and stereoselective mode

of action and the application of relatively mild reaction conditions compared to chemical

catalysis. Moreover, the mutual incorporation of the concept of enzyme and chemical

catalysis involves minimal or no implication of protecting group strategies while designing the

synthetic route due to selectivity of a particular enzyme involved. In this study, the focus has

been directed towards the application of Candida antarctica lipase B (CAL-B) catalyzed

aminolysis. Since this class of hydrolases has been established for hydrolysis and

esterification/transesterification transformations, therefore, it was considered worthwhile to

evaluate the catalytic behaviour of CAL-B towards aminolysis. Lipases, unlike proteases, do

not exhibit amidase activity75 and hence this feature makes them suitable candidates for the

synthesis of corresponding propargylamides 3 via aminolysis of methyl ester substrates 1 by

propargylamine (2). During the early optimization studies, various enzymes, such as

Candida antarctica lipase B (CAL-B), Candida antarctica lipase A (CAL-A), Candida rugosa

lipase (CRL), porcine pancreatic lipase (PPL), Pseudomonas cepacia lipase (PCL) and

protease from Bacillus licheniformis (Alcalase), have been screened for the desired

transformation and various parameters such as solvent selection, nature of ester

substrates 1, operating temperature conditions and the effect of mode of immobilization

of enzyme, have been studied. The optimization studies suggested CAL-B (Novozyme® 435)

to be the optimum biocatalyst for the desired transformation. A correlation between structural

features of ester substrates 1 with that of optimum operating temperature conditions, has

been developed that eventually led to the establishment of the control of the regioselectivity

of the action of CAL-B for selective site directed aminolysis of diester substrate 1k. Methyl

esters of N-protected amino acids (both D- and L-) have also been employed for screening

the suitability of CAL-B catalyzed aminolysis of respective amino acids that in turn instigated

the receptivity of CAL-B for such substrates since usually proteases are the enzyme of

choice for amide linkage (peptide bond) synthesis. Scheme 1.1, shows the optimized CAL-B

catalyzed aminolysis of various ester substrates 1 by propargylamine (2).

Scheme 1.1. Optimized CAL-B (Novozyme® 435) catalyzed aminolysis of various ester

substrates 1 using propargylamine (2).

Summary

6

A variety of ester substrates 1 with different aromatic and heterocyclic side chain

substituents, exhibited suitability for aminolysis by CAL-B catalysis for the corresponding

propargylamide 3 generation.

One of the hallmarks of multicomponent syntheses is the attainment of complexity in the

target molecules using relatively simple starting materials. Since the benign and selective

nature of enzyme catalysis offers the possibility to introduce various sensitive functionalities

and hence complexity in a product, therefore, the concept of incorporation of biocatalysis in

multicomponent syntheses was considered worth exploring. The terminal alkyne moiety of

propargylamides 3 obtained by CAL-B catalysis, acts as a suitable motif for further

transformations such as cycloaddition and coupling reactions, hence paving the way to the

development of one-pot synthetic schemes. The triazole ring is a bioisostere of amide

linkage70 and amide ligated 1,4-disubstituted 1,2,3-triazoles have been termed as

peptidomimetics70-71 owing to their ability to mimic the properties of peptide linkage.

Propargylamides 3 are considered to be "stubborn substrates" towards cycloaddition

reactions174 that explains the limited literature availability pertaining to the syntheses of

amide ligated triazole systems 5. The success of the previously optimized CAL-B catalyzed

propargylamide 3 formation, provided the motivation to explore the possibility of the

combination of CAL-B catalysis with Cu(I) catalyzed click reaction in a one-pot fashion. The

optimization studies for the click reaction of propargylamides 3 using conventional reaction

conditions were futile as low yields of the corresponding triazole systems 5 were obtained.

However, the application of the concept of ligand promoted click reaction conditions176a-c

furnished the higher yield of corresponding triazoles 5 under relatively mild reaction

conditions using minimum additives (Scheme 1.2).

Scheme 1.2. Chemoenzymatic one-pot synthetic scheme of amide ligated 1,4-disubstituted

1,2,3-triazoles 5.

The successful concatenation of biocatalysis (CAL-B) with the chemical catalysis, i.e. Cu(I),

further motivated the development of one-pot synthetic strategies involving other metal

catalytic systems such as palladium catalyzed Sonogashira coupling reaction of terminal

alkyne moiety of propargylamides 3. Propargylamides 3 have been reported to successfully

undergo Sonogashira coupling144,145 for the synthesis of systems containing a rigid arylated

Summary

7

propargylamide core161-160 that have established their importance as antagonists against

gonadotropin releasing hormone receptor (GnRHR)160 while propargylated deoxynucleoside

derivatives have been pursued for their application in DNA sequencing.161 One-pot

syntheses of such rigid propargylated aryl systems 6 have been successfully developed

using a variety of aryl iodides 4h-m and methyl ester substrates 1 via Pd(0)/Cu(I) catalyzed

arylation of propargylamides 3 (Scheme 1.3). Contrary to the previously reported coupling

conditions,144-145 the propargylamides 3 displayed the reactivity towards coupling under Pd(0)

catalytic conditions.

Scheme 1.3. Chemoenzymatic one-pot synthetic scheme of arylated propargylamide

systems 6.

During the development of synthetic strategy, the presence of Cu(I) catalysis in the reaction

system was further utilized by extending the strategy to triazole ligation on the coupled

products 6 (Scheme 1.4).

Summary

8

Scheme 1.4. Chemoenzymatic one-pot synthesis of triazole ligated coupled product 8.

Thus, the development of chemoenzymatic catalyzed one-pot synthetic strategies has

widened the vista of relevance of biocatalysis with metal catalytic systems and further

motivates the incorporation of other available chemical catalytic systems with enzyme

catalysis for launching novel synthetic strategies.

Introduction

9

2. Introduction

2.1. Enzyme Catalysis in Organic Solvents

The use of organic solvents is an inevitable feature in the field of organic synthesis and,

therefore, if the incorporation of the concept of biocatalysis into the one-pot synthetic

methodological strategies is intended, it is important to be certain of the activity and hence

applicability of the enzymes of interest in respective organic medium. Water is a natural

medium for enzyme action mainly responsible for maintaining its native active site

conformation via noncovalent interactions.1 In context to their application in the domain of

organic syntheses, the studies of catalytic activity of enzymes in pure anhydrous organic

solvents have been actively pursued in past decades. The replacement of bulk water with an

organic medium has been reasoned since only a few monolayers of water bound to the

enzyme's active site, are responsible for maintaining the catalytic activity1 while the retention

of catalytic activity has been rationalized on the basis of acquired structural rigidity in an

organic medium keeping the native-like conformation from unfolding.2 Other potential

advantages include a shift in thermodynamic equilibria to favor synthesis over hydrolysis,

suppression of water induced side reactions, ease of product recovery and inhibition of

microbial contamination.3

The pH memory of the ionogenic groups at the active site of the enzyme depends upon the

aqueous buffer/environment from where it has been last recovered and remains unchanged

in organic media.1 When in organic solvent, the interaction of the enzyme with the

surrounding medium might further activate or deactivate the enzyme depending upon the

nature of interactions. These interactions might lead to the disruption of the native

conformation by disordering the hydrogen bonding and other noncovalent interactions

resulting in instability.4 Solvent may also interact with diffusible components (substrates or

products) of the reaction mixture. This has been shown by the inactivation of enzyme during

peroxidase-catalyzed oxidation of phenols in chloroform since it is a quencher of phenoxy

radicals. Thereby inhibiting the overall process that requires the initiation by the enzymatic

generation of phenoxy radicals.5 The other mode of interaction depends upon the hydrophilic

nature on the organic solvent interacting with essential water bound to the active site of the

enzyme. Horseradish peroxidase catalyzed oxidation of p-anisidine in various organic

solvents showed the reduced activity of the enzyme in highly polar hydrophilic solvents

mainly due to their ability to interact with and consequently remove the essential water from

the active site of the enzyme while the highest activity was observed in relatively less polar

hydrophobic solvents.5 Various enzymes have been reported to be active in organic media

with a certain percentage of water present in it.6-9

Introduction

10

In continuation to this, porcine pancreatic lipase (PPL) catalyzed transesterification reaction

between tributyryl glycerol and various primary and secondary alcohols have been studied in

relation to water concentration in the respective organic medium that led to the finding of

retention of lipase activity at water concentration as low as 0.015 percent with the half life of

12 h in dry organic medium (decanol) at 100 °C.10 The thermal stability of PPL was shown to

have an inverse relation to the water concentration in the reaction mixture, thus establishing

the acquirement of thermal stability and altered substrate selectivity by dry enzyme in dry

organic medium. Further, in a comparative study, PPL, yeast and mold lipases have been

shown to exhibit catalytic activity towards transesterificatin, esterification, aminolysis,

thiotransesterification, and oximolysis in organic media comparable to their activity in water1

while subtilisin and α-chymotrypsin also displayed catalytic activity for transesterification

processes in organic solvents.2

2.2. Enhancement of Enzyme Activity via Excipients

The structural rigidity of proteins in non-aqueous environment is responsible for the

molecular imprinting phenomenon11 called "ligand induced enzyme memory" or

"bioimprinting".12 Ligand memory of an enzyme is directly responsible for the activating effect

of active site ligands during lyophilization.13 The structural imprint imposed by the ligand,

removed prior to the use of enzyme, on the active site of the enzyme, enables to induce the

rigid frozen conformation and readily accepts the substrates of related structural features

leading to the consequent enhanced reactivity.11,13 This approach has been employed for

alteration and enhancement of the enzyme selectivity for a given set of structurally related

substrates. The precipitation of α-chymotrypsin, which exhibited a high selectivity for L-

isomer, with n-propanol in the presence of N-acetyl-D-tryptophan, made the enzyme active

towards D-isomers, an effect absent when no precipitation with D-isomer was performed.14

Another approach for the enhancement of activity of enzyme in organic media involved the

addition of excipients or salts to the enzyme before lyophilization that prevented the

conformational changes of protein structure in organic media.15 Salts such as potassium

chloride16 and macrocyclic additives such as crown ethers,17 cyclodextrin,18,19 and polymeric

additives such as polyethylene glycol (PEG)20,21 have been shown to augment the enzyme

activity in organic medium. Salts are believed to increase the Mucor javanicus lipase and

subtilisin Carlsberg activity by increasing the polarity of the active site thereby stabilizing the

transition state.16 Cyclodextrin amplified the solubility of various substrates via host-guest

complex formation18 while crown ether worked by refolding the protein to a

thermodynamically stable catalytically active conformation via macrocyclic interactions.17

PEG activated the enzyme function by not only conserving the protein structure but also by

allowing the dissolution (upto 0.2 mg/ml) of the enzyme in organic media.20,21

Introduction

11

2.3. Stabilization of Enzymes via Immobilization

In order to increase the stability and operational efficiency of the enzymes, immobilization

techniques have been evolving ever since their development. The main idea behind the

immobilization of enzymes is to derive the obvious advantages of recycling of catalyst

system from the reaction medium and possible anticipated activity related advantages.

There is no rule to predict the resultant activity and stability of an enzyme after the

immobilization process.22 It may inhibit or increase the enzyme activity.23 Immobilized

enzymes have been shown to exhibit better thermal stability24 and enhanced resistance of

Candida rugosa lipase (CRL) against deactivation induced by aldehyde.25 Over the years,

various methods have been developed such as entrapment within a support (membrane,

gel, microcapsule), adsorption by physical interactions and cross linking techniques.26

Carrier materials such as activated charcoal,27 alumina,28 silica,29,30 celite,31 cellulose,32 and

synthetic resins,33 have been employed for the adsorption of enzymes. Adsorption has been

adopted as a method of choice when the use of lipophilic organic solvents is involved,

where desorption cannot occur.34 The efficiency of the physical adsorption of the enzyme

depends on several factors such as size of the protein to be adsorbed and carrier's specific

area and porosity, thereby controlling the rate of diffusion of substrate(s).22 Another mode of

enzyme activation is through "cross-linked enzyme aggregate (CLEA)" formation where the

individual molecules (crystals) of an enzyme get cross-linked via covalent bonding either

with each other or by using an inert filler such as albumin34 or bifunctional agents such as

glutaraldehyde, diimidates or disulfonylchloride,35 that could lead to a general enhancement

of conformational stability.36 However, cross linking impeded the enhanced catalytic activity

due to the diffusional limitation of substrate molecule(s) to the active site of the enzyme.34

2.4. Enzyme Selectivity

One of the pronounced features of enzyme catalysis is the selectivity of their mode of action

especially when heterofunctional substrates are involved. In a study, the relative reactivity of

hydroxyl and amino group towards acylation was evaluated using aminoalcohols as model

substrates without the need of protection of either of the functional groups depending upon

the nature of enzyme and acylating agent employed, thus making the chemoselective control

of the process possible.37 In another study, the substrate specificity of the transesterification

reaction catalyzed by serine protease, subtilisin Carlsberg, was shown to be strongly

dependent upon the physicochemical properties of the solvent38,39 leading to the derivation

of an enzyme-independent thermodynamic model for the prediction of substrate specificity

by any enzyme as long as the substrates are inaccessible to the solvent in transition state.39

Thus the physicochemical control of the solvent could be beneficial for inducing the desired

enantioselectivity in an enzyme.

Introduction

12

2.5. Chemoenzymatic Multicomponent Reactions

Multicomponent reactions (MCRs) are convergent reactions involving the reaction of three or

more starting materials to form the product where all or most of the atoms contribute to the

corresponding product formation.40a Hence this approach establishes high operational and

cost efficiency and atom economy of the overall process with minimum generation of by

product(s) leading to high structural diversity.40b Depending upon the sequence of addition of

reactive components, MCRs can broadly be divided into the following three categories.40b

1. Domino MCRs: in which all the components are initially present but intermediates are not

isolable.

2. Sequential MCRs: in which the components are introduced into the reaction mixture in

sequential manner under constant conditions and intermediates are isolable.

3. Consecutive MCRs: in which reaction conditions are changed in a step wise manner and

intermediates are isolable.

Since chemoenzymatic transformations have been holding primordial importance mainly

because of their ability to carry out chiral resolution of racemic or meso substrates as a

catalytic access to enantiomerically enriched chiral building blocks in organic syntheses,40c

therefore, the concept of incorporation of biocatalysis with chemical catalyzed processes has

been pursued in recent years for enhancing the structural diversity of the desired products.

Based upon the work that has been reported in past decade, the approach of incorporation

of chemoenzymatic catalysis in MCRs can clearly be divided into the following four

categories:

1. MCRs commencing with enantiomerically pure starting materials obtained by enzyme

catalyzed resolution of racemic mixtures. (not strictly a one-pot process)

2. Racemic products obtained by MCR strategy that can subsequently be submitted to

enzymatic resolution to get the optically pure product of interest. (not a one-pot process)

3. Cascade MCRs involving chemo and enzymatic catalysis running side by side. (one-pot

process)

4. Enzyme catalyzed MCRs. (one-pot process)

Introduction

13

2.5.1. MCRs Utilizing Enzymatic Resolution of Racemic Mixtures

2.5.1.1. Ugi Four-Component Reaction

Integration of enzyme catalyzed desymmetrization with Ugi four-component reaction

proceeded in a two step one-pot fashion and the yields were comparable to when conducted

in separate step wise manner, however, the one-pot process failed to establish a good

diastereoselectivity.41 With the exceptions of a few starting materials, low overall yields of

the products were obtained that were strongly dependent upon the nature of starting

materials and the nature of the co solvent for Ugi reaction (Scheme 2.1).

Scheme 2.1. One-pot integration of enzymatic desymmetrization with Ugi reaction.41

This feature of enzyme catalyzed desymmetrization has been successfully developed and

employed for desymmetrization of meso-pyrrolidine catalyzed by monoamine oxidase N

(MAO-N) from Aspergillus niger.42 Chirally pure pyrrolidines served as an entry for the

synthesis of hepatitis C virus (HCV) NS3 protease inhibitor telaprevir, in combination with

Ugi MCR,43 and further expansion of the study led to the development of a novel Ugi and

Pictet-Spengler-type cyclization for the synthesis of polycyclic 2,5-diketopiperazines (DKPs)

(Scheme 2.2).44

Introduction

14

Scheme 2.2. Oxidative desymmetrization of meso-pyrrolidine to optically pure diastereomer

for subsequent syntheses of DKP derivatives and telaprevir via Ugi-Pictet-Spengler MCR

and Ugi MCR sequence respectively.43,44

In another approach, a nine member Ugi condensation library has been developed by PPL

catalyzed acylation of 3-hydroxybutyrate and 4-amino-1-butanol with a variety of acyl

donors, selectively acylating at the hydroxyl moiety of both these precursors for the

subsequent Ugi reaction.45 In this study, the chemoselective nature (selective acylation of

hydroxyl moiety) has been exploited for generating the precursors that would subsequently

act as two of four components for Ugi reaction but the stereoselectivity was not established

(Scheme 2.3).

Scheme 2.3. Combination of PPL catalyzed acylation of carboxylic acid and amine

precursors for subsequent Ugi four-component reactions in a two step process.45

Introduction

15

In another study, enantiomerically pure chiral cyclic imine precursors, synthesized by Amano

PS lipase catalysis, have been employed for subsequent high diastereoselective

Ugi reaction for the synthesis of pharmacologically relevant polyfunctionalized pyrrolidine

systems.46 This approach exploited the enantioselectivity exerted by Amano PS lipase and

high diaseteroselectivity of Ugi reaction to achieve a full control of relative and absolute

configuration of the final product (Scheme 2.4).

Scheme 2.4. Diastereoselective Ugi reaction using chiral cyclic imine synthesized by Amano

PS lipase.46

In the previously mentioned approach,46 the concept of enzyme catalysis was employed for

the synthesis of enantiomerically pure precursors for successive Ugi reaction, however this

approach has also been envisaged for obtaining enantiomerically pure Ugi product via

enzyme mediated kinetic resolution of crude Ugi product for the synthesis of 1,3-diol

peptidomimetics.47 The lipases screened for this study, exhibited poor diastereoselectivity

and hence this approach was not pursued, however, the best result of enzymatic hydrolysis

(enantioselective approach) was obtained using Rhizopus arrhizus lipase (RAL) and

Candida antarctica lipase B (CAL-B commercially known as Novozyme® 435) with high

enantioselectivity (Scheme 2.5).

Introduction

16

Scheme 2.5. Enzymatic kinetic resolution of crude Ugi product to enantiomerically pure 1,3-

diol peptidomimetic.47

2.5.1.2. Passerini Multicomponent Reaction

A synthetic route for the synthesis of α-amino acids has been devised via wheat germ lipase

catalyzed hydrolysis of α-acetoxyamides, prepared via Passerini reaction, to the

corresponding enantiomerically enriched α-hydroxyamides that, in turn, are converted to α-

aminoamides. The α-aminoamides so produced were then converted to α-amino acids

(Scheme 2.6).48

Scheme 2.6. Enzymatic resolution of Passerini product for the synthesis of enantiomerically

pure α-amino acid.48

Further investigation revealed the enantioselectivity of the wheat germ lipase to be strongly

dependent upon the hydrophobicity of the solvent employed49 and led to the synthesis of N-

Introduction

17

methylated amino acids.50 This method facilitated the introduction of diverse amino acid

moieties to the tripeptide scaffold,50 leading to the synthesis of unnatural peptides51 that

have been difficult to synthesize with high stereocontrol of the chiral centres via Ugi pathway

(Scheme 2.7).52

Scheme 2.7. Stereocontrolled synthesis of tripeptides by chemoenzymatic Passerini

reaction.51

The synthesis of α,α-dialkyl-α-hydroxycarboxylic acid derivatives have been achieved via

hydrolase catalyzed kinetic resolution of the racemic mixture obtained by Passerini

reaction.53 Out of 40 screened enzymes, a protease (P1), a thermophilic esterase (PestE),

and an esterase of metagenome origin (esterase 8) were the most active and

enantioselective (Scheme 2.8).

Scheme 2.8. Synthesis of N-(t-butyl)-2-hydroxy-2,2-dialkylamides by Passerini MCR and

subsequent enzymatic kinetic resolution.53

Introduction

18

2.5.1.3. Biginelli Multicomponent Reaction

The preparation of enantiomerically pure dihydropyrimidones (DHPMs) obtained by Biginelli

reaction, has been achieved by lipase catalyzed resolution of activated DHPM esters but the

overall process exhibited lower selectivity when conducted on analytical scale.54 Activated

DHMP derivative with an acetoxymethyl residue at N-3 position, underwent selective

cleavage by Thermomyces lanuginosus lipase (Amano CE lipase) for the synthesis of

optically pure enantiomers. Further derivatization of (R)-enantiomer furnished

antihypertensive agent (R)-SQ 32926 (Scheme 2.9).

Scheme 2.9. Lipase catalyzed resolution of DHMPs (Biginelli product) for the subsequent

synthesis of antihypertensive agent SQ 32926.54

2.5.1.4. Synthesis of Cyclic Systems

4-N-Phenylacetylamino derivatives obtained by three-component coupling of anhydrides,

phenylacetamide and dienophiles (ANAD sequence) were subjected to kinetic resolution via

hydrolysis conducted by Penicillin G amidase (PGA) from Escherichia coli.55 Moreover, in

silico predictions based upon the simulation of enzyme-substrate interaction, were found to

be in agreement with the observed selectivity of the enzyme.55 Further study involved the

application of a number of different hydrolases such as Chirazyme® E-3, Burkholderia

cepacia lipase (BCL), CAL-B (Novozyme® 450), PGA-450 (immobilized PGA), and p-

nitrobenzyl esterase (p-NBE), to improve the enantioselectivity of the process (Scheme

2.10).56

Introduction

19

Scheme 2.10. Kinetic resolution of ANAD sequence products mediated by hydrolases.56

The kinetic resolution of racemic mixture of the 3-azabicyclo[3.2.0]heptane derivatives,

synthesized by a multicomponent cascade reaction (combination of aza-Michael addition,

intramolecular Michael addition and Mannich-type reaction), mediated by CAL-B

(Novozyme® 450), furnished enantiopure derivatives that exhibited enhanced activity as

dopaminergic ligands (Scheme 2.11).57

Scheme 2.11. Chemoenzymatic synthesis of dopaminergic ligand 3-aza-bicyclo[3.2.0]

heptane.57

Introduction

20

The synthesis of 6,7-dihydrobenzofuran-4(5H)-ones has been accomplished by combining

laccase-catalyzed formation of (Z)-3-hexene-2,5-dione by oxidative cleavage of 2,5-

dimethylfuran that subsequently underwent Lewis acid catalyzed domino 1,2-addition/1,4-

addition/elimination with 1,3-dicarbonyl compounds in one-pot fashion (Scheme 2.12).58

Scheme 2.12. Chemoenzymatic one-pot synthesis of 6,7-dihydrobenzofuran-4(5H)-ones.58

2.5.1.5. Strecker Reaction

The development of an environmentally benign robust double dynamic asymmetric Strecker

multicomponent system coupled with kinetically controlled Pseudomonas cepacia lipase

(PCL) mediated transacylation facilitated the synthesis of enantiomerically pure acylated-N-

substituted α-aminonitriles (Scheme 2.13).59

Scheme 2.13. One-pot PCL mediated kinetic resolution of α-aminonitriles obtained by

Strecker reaction under thermodynamically controlled double dynamic covalent system.59

2.5.2. Enzyme Catalyzed Multicomponent Reactions

The promiscuity of enzyme action has been a topic of interest for their effective application

and eventual design of multicomponent reactions. Thus this led to the development of

enzyme catalyzed multicomponent Ugi, Hantzsch, Mannich, Biginelli, and some novel

reaction sequences thus showcasing the aptness and capacity of biocatalysis for

multicomponent reactions.

Baker's yeast (Saccharomyces cerevisiae) has been reported to synthesize dihydropyridyl

derivatives via Hantzsch reaction.60 This aspect of catalysis of Baker's yeast was further

explored for the synthesis of aryl substituted polyhydroquinoline derivatives via

unsymmetrical Hantzsch reaction (Scheme 2.14).61

Introduction

21

Scheme 2.14. Baker's yeast catalyzed four-component unsymmetrical Hantzsch reaction.61

Later, the synthesis of 3,4-dihydropyrimidin-2-(1H)-ones was conducted via Biginelli reaction

catalyzed by Baker's yeast (Saccharomyces cerevisiae).62 The developed methodology

represented a set of relatively mild reaction conditions for introducing functional group

diversity in the resultant product (Scheme 2.15).

Scheme 2.15. Baker's yeast catalyzed Biginelli reaction for the synthesis of 3,4-

dihydropyrimidin-2-(1H)-ones.62

An inexpensive, environmentally benign waste free methodology was developed for Biginelli

reaction using bovine serum albumin (BSA) and the practicality of the methodology was

demonstrated by recyclability of the catalyst system (BSA) on gram-scale synthesis of the

corresponding bioactive derivative monastrol (Scheme 2.16).63

Scheme 2.16. Gram-scale synthesis of monastrol by Biginelli reaction catalyzed by bovine

serum albumin (BSA).63

Lipase from Mucor miehei (MML) has been effectively employed for Mannich reaction in

water for the synthesis of β-amino ketone derivatives (Scheme 2.17).64

Introduction

22

Scheme 2.17. MML catalyzed Mannich reaction for the synthesis of β-amino ketone

derivatives.64

Since one of the reacting species was acetone that also served as co solvent with water but

was not considered appropriate for the design of environmentally benign synthesis and also

because of the competing reaction of acetone with other ketones to be employed,65

therefore, further studies in same direction using a variety of substrates, used EtOH:water

mixture as an environmentally benign solvent system and Candida rugosa lipase (CRL) as

the enzyme of choice to achieve a successful conversion to the corresponding structurally

diverse β-amino ketone derivatives.65

First model reaction for the synthesis of dipeptide via Ugi three-component reaction has

further established the suitability of CAL-B (Novozyme® 450) for such multicomponent

reactions and expanded the scope of the study by demonstrating its efficacy in aqueous

medium. However aqueous medium has not been considered suitable for Ugi reaction due

to the susceptibility of the decomposition of isocyanide in the presence of acid (Scheme

2.18).66

Scheme 2.18. Dipeptide synthesis via Candida antarctica lipase B (Novozyme® 435)

catalyzed Ugi reaction.66

The development of PPL-catalyzed novel one-pot four-component reaction furnished a 2,4-

disubstitued thiazole system under milder reaction conditions thus showcasing the suitability

of biocatalysis for the synthesis of heterocyclic systems (Scheme 2.19).67

Introduction

23

Scheme 2.19. PPL catalyzed novel four-component multicomponent reaction for the

synthesis of 2,4-disubstituted thiazole synthesis.67

Spirooxazino derivatives have been synthesized by a novel CAL-B (Novozyme® 435)

catalyzed multicomponent reaction from readily available starting materials furnishing six

new C-C/-N bonds and two ring systems in a single step (Scheme 2.20).68

Scheme 2.20. CAL-B (Novozyme® 435) catalyzed novel multicomponent reaction for the

synthesis of spirooxazino derivatives.68

In context to the physiological and pathological significance of sialic acid derivatives, a

number of respective derivatives has been generated via one-pot three enzyme system

(Scheme 2.21).69

Introduction

24

Scheme 2.21. Synthesis of α-2,6-linked sialosides via one-pot three enzyme system.69

Aim of the Project

25

3. Aim of the Project

In context to the applicability of enzyme catalysis for organic transformations, it was

considered worthwhile to incorporate this concept with chemical catalysis for the

development of new multicomponent one-pot strategies. The entry to the multicomponent

sequence has been designed to be comprised of lipase catalyzed aminolysis of methyl ester

substrates 1 for the synthesis of the corresponding propargylamides 3 that, in turn, would act

as motif for further transformations to cycloadded (1,4-disubstituted 1,2,3-triazoles 5),

coupled (arylated propargylamides 6) and cycloisomerized products (oxazoles 7), owing to

the presence of a terminal alkyne moiety. The intended cycloaddition (Cu(I) catalyzed click

reaction) would give rise to the corresponding amide ligated 1,4-disubstituted 1,2,3-triazole

systems 5. The triazole nucleus has been established as bioisostere of amide linkage that

mimics the atom placement and electronic properties of peptide bond and exhibits enhanced

stability due to its resistance towards hydrolytic cleavage70,71 thus paving the way for amide

ligated triazole systems as interesting peptidomimetic motif, to drug development since,

unlike amide linkage, triazole nucleus is not susceptible to proteolytic cleavage.71,72

Moreover, acetylene derived amino acid chains can further be ligated to peptide sequences

for labelling or conjugation purposes.71 Keeping in view the established antitubuline and anti

tumor activity of oxazoles,73 coupling and subsequent cycloisomerization of propargyl-

amides 3 has been intended that would give rise to corresponding 2,5-disubstituted oxazoles

7 with possible anticipated biological activities.

The main challenges have been anticipated to be the choice of an appropriate biocatalyst for

the desired transformation, solvent selection, synthesis of suitable ester substrates 1 and

ultimately, the compatibility, optimization and tunability of biocatalyzed process with the

subsequent chemical catalysis (Cu(I) and Pd) for developing viable one-pot processes. A

brief outline of the research idea has been sketched as a retrosynthetic analysis in Scheme

3.1.

Aim of the Project

26

Scheme 3.1. Intended multicomponent one-pot synthetic strategies for the synthesis of

amide ligated 1,4-disubstituted 1,2,3-triazole 5 via Cu(I) catalyzed click reaction and the

synthesis of coupled product 6 via Sonogashira coupling and its subsequent

cycloisomerization to oxazole systems 7 commencing with lipase catalyzed aminolysis of

ester substrates 1 by propargylamine (2).

Literature Review

27

4. Literature Review

4.1. Mechanistic Aspects of Lipase Catalyzed Aminolysis

Lipases catalyzed aminolysis of ester substrate with a nucleophile, follows "serine hydrolase

mechanism".74 The mechanism involves the activation of an ester substrate by the formation

of an acyl-enzyme complex at the active site that makes the acyl moiety of the ester

susceptible for the attack by an appropriate nucleophile.34 The histidine residue, activated by

the aspartate side chain,74 is in proximity to the serine residue at the active site of the

enzyme and this arrangement results in its lower pKa value,34 making it a suitable

nucleophile for the attack at the carbonyl moiety of ester substrate leading to the covalent

bonded acyl-enzyme intermediate. Thus this acyl-enzyme intermediate, in the presence of

an appropriate nucleophile, regenerates the enzyme and liberates the corresponding product

from the active site (Scheme 4.1).

Scheme 4.1. Formation of an acyl-enzyme intermediate at the active site of the enzyme

followed by the attack of an appropriate nucleophile for the generation of corresponding

product and regeneration of the native conformation of the enzyme.74,34

Literature Review

28

Depending upon the nature of the nucleophile, the acyl-enzyme complex may give rise to the

corresponding products (Scheme 4.2).

Scheme 4.2. Formation of different products arising from the attack by a nucleophile on the

acyl-enzyme complex.34,74

Thus this mechanistic feature of serine hydrolases has been actively exploited for organic

synthesis in pure organic medium where, under water free conditions, other nucleophiles

can equally be compatible for carrying out the respective transformations.74

4.2. Development of Lipase Catalyzed Aminolysis

Proteases have been the enzyme of choice for the generation of amide linkage in peptide

synthesis however their utility for such transformation has been limited by their amidase

activity, preference towards L-configuration of amino acids, and stability issue in anhydrous

organic solvents.75 These limitations prompted the exploration of possible application of

esterases and lipases for the synthesis of peptides and hence led to the study addressing

the potential of porcine pancreatic lipase (PPL), Candida cylindracea lipase (CCL), and pig

liver esterase (PLE) for peptide synthesis containing usual and unusual amino acids in the

sequence.75 Furthermore, the study of CCL for enantioselective aminolysis of the racemic

mixture of ethyl 2-chloropropionate with several aromatic and aliphatic amines for

corresponding chiral amide syntheses, established their suitability for the respective

transformation even when employed below room temperature.76 Enantioselective acylation of

amino alcohols for corresponding pharmaceutically enantiopure chiral amide synthesis

catalyzed by PPL established its efficacy compared to the available relatively expensive

chemical method.77 Propargylamide synthesis has been reported using CCL to catalyze the

reaction between ethyl propiolate and aromatic amines78 since aliphatic amines mainly tend

to give the Michael addition product (Scheme 4.3).79

Literature Review

29

Scheme 4.3. CCL catalyzed propargylamides using ethyl propiolate and aromatic amines.78

Double aminolysis of racemic ethyl 2-chloropropionate catalyzed by CCL resulted in the

formation of the desired (S,S) diester in high enantiomeric excess compared to Amano P

lipase and subtilisin that exhibited poor enantioselectivity.79 Moreover, the overall

transformation to the diester was strongly dependent not only upon the enzyme but also the

solvent employed, CCl4 and diisopropyl ether being the most suitable ones. Further

elaboration of the study pertaining to (S)-enantioselective monoamidation of racemic ethyl 2-

chloropropionate catalyzed by CCL, displayed the crucial role played by the solvent

selection, nonpolar solvents (n-hexane and CCl4) being the most suitable ones.80 This also

led to the finding of the reversal of role, enantioselectivity wise, of CCL exhibited (R)-

enantioselectivity in transesterification reactions.81 The study of the amidation reaction of

racemic ethyl 2-chloropropionate with racemic aliphatic amines showed the preference of

CCL for (S)-enantiomer of the ester while subtilisin preferred the (S)-enantiomer of the

amine but no absolute selectivity was observed by the use of either enzymes towards ester

and amine concurrently.82

RAL have been studied to show high (R)-enantioselectivity for the aminolysis of palmitic acid

with racemic 2-amino octane compared to CCL, PPL, and Penicilium cyclopium lipase.83

Solvent free aminolysis of ethyl butyrate with various aliphatic and aromatic amines

catalyzed by lipase SP 382 (mixture of CAL-A and CAL-B) illustrated the dependence of the

reaction strongly upon the structural features of the amines employed.84 Aliphatic primary

amines were more reactive than secondary ones while tertiary amines did not show any

receptivity by the enzyme and the aminolysis by aromatic amines produced lower yields of

the corresponding amide products. The access to enantiomerically pure 1,3-aminoalcohols

was made possible by the enantioselective aminolysis of racemic ethyl 3-hydroxybutyrate

effectively catalyzed by CAL compared to PCL, that then underwent subsequent reduction

by LiAlH4 with little or no racemization.85,86 No reactivity was observed when aromatic amines

were employed, however, highest enantioselectivity was observed using benzylamine

(Scheme 4.4).

Literature Review

30

Scheme 4.4. CAL catalyzed synthesis of 3-hydroxyamides and their subsequent conversion

to enantiomerically pure 1,3-aminoalcohols.85,86

PPL exhibited high enantiospecificity when N-benzyloxycarbony (Cbz) protected amino

alcohols have been used compared to the nonprotected substrates. PPL was more efficient

in catalytic activity compared to CCL and CAL.87 CCL and CAL have been screened for their

suitability for chiral amide formation using activated (having an electron withdrawing group at

the α-position) and nonactivated ester substrates.88 The (S)-enantioselectivity displayed by

CCL in earlier studies,2,6 has been shown to be independent of the size of the amine while

CAL was selective towards the (R)-enantiomer with moderate enantiomeric excess of the

corresponding products. CAL has been employed as the enzyme of choice for aminolysis

and ammonolysis of β-ketoesters for the generation of β-ketoamides (Scheme 4.5).89

Scheme 4.5. CAL catalyzed aminolysis/ammonolysis of β-ketoesters.89,90

With racemic amines, CAL catalyzed the aminolysis of the (R)-enantiomer, generating the

corresponding β-ketoamide in high enantiomeric excess.90 Moreover, the ability of CAL-B

was explored for a mild and practical enantioselective synthesis of the chiral amides by the

ammonolysis of carboxylic acids.91 This feature of CAL-B catalysis was further explored and

effectively employed for their dual role for the esterification of octanoic acid with ethanol

followed by ammonolysis to furnish octanamide.92 A solvent free environmentally benign

protocol for the synthesis of chiral amides has been devised using carboxylic acids as acyl

Literature Review

31

substrate and racemic amines catalyzed by CAL-B that exhibited its (R)-enantiopreference

for the amines employed.93

The synthesis of the corresponding amides by aminolysis of nonactivated esters with β-furyl

and β-phenyl groups has been achieved using CAL as the enzyme of choice.94 Additionally,

the aminolysis with the chiral amines established (R)-enantioselectivity of CAL with high

enantiomeric excess values. The ester substrates with multiple bonds such as propiolic and

acrylic esters, have also been converted to the corresponding amides using CAL as enzyme

of choice.95 The choice of solvent was crucial in the suppression of competing Michael 1,4-

addition formation. The results of aminolysis with racemic amines was consistent with the

previously established (R)-enantioselectivity of CAL.94 The study of suitability of CAL-B for

α,β-unsaturated esters led to a selective propiolamide synthesis without the formation of

competing Michael adduct formation using immobilized CAL-B.96 Following the same line of

argument, chemoselective control favoring 1,2-addition (amide formation) in a reaction

between aliphatic amines and ethyl propiolate was further studied. The optimum conditions

for such chemoselectivity involved the use of immobilized CAL-B (Novozyme® 435) in

solvents such as toluene, 1,4-dioxane, and MTBE (Scheme 4.6).97

Scheme 4.6. Chemoselective amide formation (1,2-addition) catalyzed by CAL-B using ethyl

propiolate and amines.96

In order to overcome the difficulties encountered during the chemical procedure of

alkoxycarbonylation for chiral carbamate synthesis, the practicality of CAL for such

transformation was explored leading to successful generation of corresponding chiral

carbamates, with a few exceptions, generally retaining (R)-enantioselectivity that strongly

depended upon the nature of substrates and solvent employed (Scheme 4.7).98

Scheme 4.7. CAL catalyzed alkoxycarbonylation transformation.98

Literature Review

32

The reversal of enantioselectivity was observed when racemic vinyl carbonates were

employed, in which case CAL exhibited (S)-enantioselectivity for vinyl carbonates (Scheme

4.8).99

Scheme 4.8. CAL catalyzed kinetic resolution of vinyl carbonates exhibiting (S)-

enantioselectivity for carbonates.99

Alkoxycarbonylation of the primary amino group of pyrimidine 3,3-diaminonuceloside has

been performed using CAL-B and homocarbonates for the synthesis of corresponding N-5-

carbamates (Scheme 4.9).100

Scheme 4.9. CAL-B catalyzed alkoxycarbonylation of 3,5-diaminonucleoside using

homocarbonates. 100

The results of systematic study of the variables controlling the action of CAL-A and CAL-B

towards ammonolysis, demonstrated that both isoforms of lipase from Candida antarctica,

CAL-A (SP 526) and CAL-B (SP 525), exhibited (R)-enantiopreference in the aminolysis of

an acyl donor.101 The enantiomeric purity of the amides obtained, established the higher

stereoselectivity of CAL-B compared to CAL-A and immobilization of CAL-B on Lewatit® E

(Novozyme® 435) had no effect on (R)-enantiopreference.

The mono ammonolysis and aminolysis of dimethyl succinate with ammonia and aliphatic

amines respectively, have been conducted using CAL-B as the enzyme of choice that also

furnished optically active amidoesters when racemic amines were used.102 Further studies

employing diamines with dimethyl succinate and dimethyl glutarate furnished polymeric

materials such as N-N-polymethylenesuccinimides and N,N-polymethyleneglutarimides

catalyzed by CAL.103 However, in both studies,102,103 the formation of cyclic imides has been

observed along with other desired products (Scheme 4.10).

Literature Review

33

Scheme 4.10. Cyclic imide formation by CAL via cyclization involving the less hindered

nitrogen atom.103

The regioselective action of CAL upon diester substrates was further explored when Cbz-

protected glutamic diesters were used.104 The results of the study showed that Cbz-L-Glu

diester regioselectively underwent α-amide formation while the D-enantiomer furnished γ-

amide. CAL exhibited (R)-enantioselectivity for the chiral amines used.

Pseudomonas cepacia lipase (Amano P-30) has been shown to catalyze aminolysis of

benzyl ester with a variety of different amines.105 Further, the aminolysis of α-hydroxy benzyl

ester with different amines furnished α-hydroxyamides with high stereoselectivity.106 The

enhanced catalytic activity of Amano P-30 lipase towards the aminolysis of esters with

oxygenated linkers with both symmetric and unsymmetric diamines107 showcased the

improved adequacy of such substrates that was mainly attributed to the inductive effect of

the oxygen atom at the α-position, making the acyl moiety of such substrates more

electrophilic and hence susceptible for enzyme attack.108 However, the enhancement of

enzyme action was observed for aminolysis, but opposite effect was observed for hydrolysis

when both the processes were carried out in a one-pot fashion (Scheme 4.11).

Scheme 4.11. One-pot aminolysis and hydrolysis catalyzed by PCL showing the selective

amide formation at the ester group with oxygenated side linker while hydrolysis occurred at

nonactivated ester group.108

The formation of lactams by intramolecular cyclization of amino esters and macrocyclic

bislactams from diesters and diamines, has been reported to be catalyzed by PPL in organic

media.109 Lower yields (40 %) of bislactams were obtained and the process was more

selective depending upon the nature of diesters and diamines employed (Scheme 4.12).

Literature Review

34

Scheme 4.12. PPL catalyzed intramolecular cyclization of amino esters to lactams.109

The ability of lipases to catalyze intramolecular cyclization of aminocarboxylic acids to

lactams was further investigated and results revealed the suitability of CAL-B (Novozyme®

435) for the synthesis of ε-caprolacton. This methodology led to the synthesis of different

sized lactam rings (4-8 membered).110

The failure of CAL-B to catalyze the corresponding ring system using mixtures of

aminocarboxylic acids, showed the preference of the enzyme for homocyclization (Scheme

4.13).

Scheme 4.13. CAL-B catalyzed synthesis of lactams.110

CAL-B (Chirazyme® L-2) catalyzed the transamidation of nonactivated amides with amines.

The study suggested the crucial role of the size of the side chain of the amide as bulky

amide substrates and aromatic amides failed to undergo transamidation while the structural

variation of the side chain of primary amines did not substantially improve the process. The

incomplete conversion was mainly attributed to the attainment of thermodynamic equilibrium

between substrates and the product (Scheme 4.14).111

Scheme 4.14. Transamidation catalyzed by CAL-B (Chirazyme® L-2).111

Lactams with carboxylic acid/ester substituents such as both (R)- and (S)-enantiomers of

pyroglutamic acids have been transformed to corresponding amide derivatives catalyzed by

CAL-B (Novozyme® 435 ) using ammonia and different primary amines whereas secondary

amines failed to furnish any amide product.112 CAL-B (Novozyme® 435 and Chirazyme® L-2)

exhibited the acceptability of bicyclic systems such as binaphthyl ester for their resolution via

aminolysis of ethylene spacer between ester functionality and the naphthyl ring (Scheme

4.15).113

Literature Review

35

Scheme 4.15. CAL-B (Novozyme® 435) mediated kinetic resolution of binaphthyl system.113

The potential of kinetic resolution carried out by lipases has been exploited for the synthesis

of enantiomerically pure starting materials for the subsequent chemical transformation to

industrially and pharmaceutically interesting compounds. The synthesis of the

enantiomerically pure aromatase inhibitor 1-(2-benzofuranyl(4-chlorophenyl)methyl)-1H-

imidazole has been carried out by CAL-B (Novozyme® 435) catalyzed kinetic resolution of 1-

(4-chlorophenyl)-2-propynylamine that served as starting material for the desired

transformation (Scheme 4.16).114

Scheme 4.16. CAL-B catalyzed kinetic resolution of 1-(4-chlorophenyl)-2-propynylamine for

subsequent chemical transformation to biologically active imidazole system.114

Pyrrolidin-3-ol is a precursor for the synthesis of alkaloids and their derivatives. The

syntheses of enantiomerically pure derivatives have been conducted by CAL-B (Novozyme®

435) catalyzed ammonolysis of racemic ethyl 4-chloro-3-hydroxybutanone, that established

the crucial role of solvent in controlling the enantioselectivity of the enzyme (Scheme

4.17).115 Starting with a racemic mixture, selective ammonolysis of the (S)-enantiomer took

place when 1,4-dioxane was used as solvent, leaving (R)-enantiomer as unreacted ester

with high ee values. The (R)-enantiomer was later transformed to corresponding amide by

ammonolysis using the same biocatalyst (CAL-B) in t-BuOH.

Literature Review

36

Scheme 4.17. Solvent dependent chemoenzymatic synthesis of pyrrolidin-3-ol.115

A two step, CAL-A (Chirazyme® L-5) and CAL-B (Novozyme® 435), catalyzed procedure has

been adopted for the synthesis of pharmaceutically interesting β-dipeptides (Scheme

4.18).116

Scheme 4.18. CAL-A and CAL-B catalyzed β-dipeptide synthesis.116

CAL-B (Chirazyme® L-2) has been successfully employed for the selective synthesis of the

secondary amide surfactant N-methyl lauroylethanolamide from methyl laurate and N-

methylethanol amine. The enzyme remained active upto six catalytic runs, establishing the

role of CAL as suitable catalyst for surfactant generation under benign and mild reaction

conditions.117 To maintain the green protocol of the process, in recent years, the suitability of

CAL-B towards amide formation has been demonstrated using ionic liquids such as

[BMIm(BF4)]118 and [BMIm(PF6)],

118,117 for the synthesis of amides with high

enantioselectivity118 with the retention of activity of the enzyme upto four consecutive

cycles.119

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4.3. Development of Synergy of Biocatalysis with Click Multicomponent Synthesis of

Triazoles

Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC), commonly known as "click reaction",

holds its importance mainly in the context of regioselective perfection of thermal Huisgen

1,3-dipolar cycloaddition, giving rise to the selective formation of 1,4-disubstituted 1,2,3-

triazole systems (Scheme 4.19).120

Scheme 4.19. a) Formation of mixture of products by thermal Huisgen 1,3-dipolar

cycloaddition. b) Cu(I) catalyzed regioselective formation of 1,4-disubstituted isomer.

In the uncatalyzed process, the alkyne has been shown to remain a poor dipolarophile.120

DFT calculations of the copper catalyzed process described the catalysis to be mediated by

Cu(I) species and the rate enhancement due to the lowering of activation energy, was

reasoned to be due to stepwise process contrary to the thermal uncatalyzed concerted

pathway.121 The reaction pathway commences with the coordination of Cu(I) species to the

π-system of alkyne thereby lowering the pKa value of the acetylenic proton resulting in the

exothermic formation of acetylide. The coordination of the azide with Cu(I) acetylide is

followed by a rearrangement to a six membered metallocycle (Va, Vb) that further transforms

into copper metallated triazole (VI).122 The copper-triazole complex (VI) then liberates the

free triazole (VII) and LnCu(I) species via protonation or reaction with another electrophile.

The kinetic measurements were not in agreement with the mechanism and indicated the

involvement of more than two copper atoms coordinated with the terminal carbon atom of

acetylene in the transition state.123 The Cambridge Crystal Database suggested each C-C

bond to be coordinated with three (90 % of all Cu(I)-alkyne complexes) or at least two

copper atoms and this type of coordination has been established to be energetically favored

for acetylide, making the secondary carbon atom more electrophilic.122 Further DFT

calculations suggested the acetylide and azide not to be coordinated to the same copper

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atom in the transition state.70,123,124 Scheme 4.20, shows a summarized depiction of the

proposed mechanisms.

Scheme 4.20. Proposed mechanism for the Cu(I) catalyzed reaction between azides and

terminal alkynes.122

Triazole ring containing derivatives have been studied to possess anti-HIV,125

antibacterial,126 anticancer,127 antifungal128 activities and have found an immense importance

in the field of material sciences,129 supramolecular assemblages,130 and combinatorial drug

discovery.130 Plethora of literature is available pertaining to the role of click chemistry for drug

development.132 However only a limited amount of literature is available corresponding to the

application of multicomponent chemoenzymatic synthesis of triazoles via click chemistry in a

one-pot fashion.

Like other multicomponent reactions, the concept of chemoenzymatic-click sequence has

been pursued for the syntheses of enantiomerically pure target compounds via enzyme

catalyzed enantioselective preparation of optically pure starting materials. In one of such

approaches, enzymatic preparation of bromoazidoconduritol has been reported via whole

cell fermentation of bromobenzene with Pseudomonas putida. After further protection of diol

and derivatization, bromoazidoconduritol was obtained and served as a substrate for

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39

subsequent palladium catalyzed coupling and click transformations, optimized both in

tandem and sequential manner (Scheme 4.21).133

Scheme 4.21. Chemoenzymatic preparation of bromoazidoconduritol for subsequent

sequential one-pot coupling-click sequence.133

One-pot sequences have been developed that involved the intermediacy of epoxide with

subsequent derivatization to azide via ring opening. This mutual compatibility displayed by

epoxide ring opening and click reaction in a one-pot process led to the development of a

tandem chemoenzymatic-click sequence for the synthesis of enantiomerically pure

regioselective chiral hydroxy triazoles. Starting off with racemic epoxide mixtures, it was

possible to carry out the enatioselective azidolysis catalyzed by halohydrin dehalogenase

(HheC) to give rise to 1,2-azido alcohols. The attack of azide primarily took place at the non-

substituted carbon atom of the ring (β-regioselectivity). The azido alcohol so formed further

participated in click reaction giving rise to chiral hydroxy triazoles.134 This methodology

accounts for an elegant eco friendly procedure for the synthesis of optically pure chiral

derivatives (Scheme 4.22).

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Scheme 4.22. One-pot chemoenzymatic-click sequence for the synthesis of chiral hydroxy

triazoles.134

The concept of convergent one-pot synthesis involving reduction by alcohol dehydrogenase

(ADH) and Cu(I) catalyzed click reaction was successful in synthesizing chiral 1,4-

disubstituted 1,2,3-triazole derived diols via intermediacy of chiral alcohols in high yields and

excellent enantio- and diastereoselectivities, showing the mutual compatibility of enzymatic

and chemical catalysis (Scheme 4.23).135

Scheme 4.23. Chemoenzymatic one-pot protocol to synthesize enantioenriched 1,2,3-

triazole derived diols.135

Systems containing a chiral alcohol moiety may be anticipated as important precursors for

the synthesis of chiral auxiliaries. In this context, a sequential two step one-pot procedure

was designed taking advantage of the synergy between the enantioselective reduction of

azidoketones by Daucus carota (carrot root) to azidoalcohols followed by triazole synthesis

yielding the corresponding chiral hydroxy disubstituted 1,2,3-triazoles (Scheme 4.24). This

chemoenzymatic sequence encompassed the obvious advantages of using cheap and

readily available starting materials in water as a green solvent.136

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Scheme 4.24. One-pot synthesis of chiral 1,2,3-triazoles from azidoacetophenones.136

A simultaneous chemoenzymatic one-pot sequence has been developed by combining click

reaction and CAL-B (Novozyme® 435) catalyzed transesterification, coupled with an atom

transfer radical polymerization (ATRP) giving rise to multicomponent polymerization (MCP)

for a tricomponent polymer system (Scheme 4.25).137

Scheme 4.25. Click–chemoenzymatic–ATRP system in one-pot to prepare designable

functional polymers.137

4.4. Synergy of Biocatalysis with Palladium Catalyzed Coupling Reactions

The Sonogashira coupling reaction has developed its efficacy as one of the most widely

employed C(sp2)-C(sp) coupling methods between aryl, alkenyl, acyl halides or triflates and

terminal alkynes for the synthesis of corresponding arylalkynes, alkenylynes and ynones138

respectively, catalyzed by Pd(0)/Pd(II) phosphane complexes and Cu(I) as co catalyst in the

presence of an appropriate base (Scheme 4.26).139 The relative reactivities of sp2 species for

Sonogashira coupling reaction follow the order: vinyl iodide > vinyl triflate > vinyl bromide >

vinyl chloride > aryl iodide > aryl triflate > aryl bromide >> aryl chloride. This methodology

has been immensely employed in the fields of dyes, sensors, electronics, polymers, natural

product chemistry and heterocycle synthesis.139b,c

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Scheme 4.26. An overview of the attainment of product diversity utilizing Sonogashira

coupling.

Despite of its extensive application in the field of organic synthesis, the exact mechanism of

the Sonogashira coupling reaction remained an issue of debate mainly due to the

involvement of two independent metal catalytic cycles (Pd(0) and Cu(I)) and the difficulty to

isolate the organometallic intermediates from homogeneous mixture to confirm several

mechanistic aspects.139c However, a concise description of the proposed mechanism is

depicted in Scheme 4.27. The mechanism is comprised of two catalytic cycles. The

palladium catalyzed cycle commences with active Pd(0)L2 species that can be formed from

Pd(0) catalyst such as Pd(PPh3)4 or Pd(II) (PdX2(Ln)2 such as PdCl2(PPh3)2) species via

coordination with terminal alkyne that then regenerates the active Pd(0)L2 species after

reductive elimination.140 Since in present work Sonogashira coupling of aryl halides 4h-4p

with propargylamides 3 has been performed, therefore, for this purpose the consideration of

energy barrier of oxidative addition, which is a rate limiting step of the process, has been

considered important that follows the order ArI < ArBr < ArCl.141 After transmetalation with

copper acetylide, generated in Cu(I) catalyzed cycle, the resulting adduct undergoes

cis/trans isomerization and then reductive elimination, generating active Pd(0)L2 species

again (Scheme 4.27). The nature of Cu(I) catalytic cycle is not well developed mainly due to

possibility of copper-ligand interaction(s) and inter transfer from one metal to other in tandem

palladium/copper coupling cycle.142

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Scheme 4.27. Proposed mechanistic involvement of two separate metal catalytic cycles

(Pd0 and Cu(I)) for Sonogashira coupling.

Due to the synthetic magnanimity of palladium catalyzed cross couplings, the concept of

incorporation of Sonogashira coupling with lipase catalyzed transformations can very well be

apprehended and anticipated to further increase the synthetic potential of the overall

chemoenzymatic process when conducted in tandem or consecutive manner. This

synergistic behaviour has been reported by carrying out lipase mediated aminolysis with

palladium catalyzed couplings (Buchwald-Hartwig, Suzuki, Sonogashira) simultaneously on

the same substrate leading to the development of a lipase-Pd cascade reaction sequence.143

The efficiency of the developed cascade reactions strongly depended upon the nature of Pd

catalyst, alcohol side product and optimization of the cascade process as a whole contrary to

the generally followed approach of stepwise optimization strategies (Scheme 4.28).

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Scheme 4.28. Study of the synergistic effect of lipase-palladium cascade reactions: a)

Buchwald-Hartwig Coupling. b) Suzuki Coupling. c) Sonogashira Coupling.143

4.5. Heterocyclization of Propargylamides

4.5.1. Oxazole Syntheses

Synthesis of the biologically active 2,5-disubstituted oxazole structure has been achieved via

sequential coupling and cycloisomerization of N-propargylamides.144 Formation of the

oxazole system was reasoned to take place following the coupling step. The subtlety and

control of reaction conditions were crucial. The coupling step was promoted by the

implementation of electron withdrawing phosphane ligands while cycloisomerization was

induced by the choice of an appropriate base and base to alkyne ratio, since excess of base

could also promote the intramolecular cycloisomerization of the propargylamide without

undergoing any coupling reaction (Scheme 4.29).

Scheme 4.29. Preparation of 2,5-disubstituted oxazole system by sequential coupling-

cycloisomerization sequence.144

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The propensity of propargylamides to undergo cycloisomerization to the oxazole ring was

further exploited and led to the development of a consecutive three-component one-pot

oxazole synthesis via an amidation-coupling-cycloisomerization (ACCI) sequence.145 The

cycloisomerization of acylated propargylamide was mediated by PTSA unlike the previously

reported method in which silica gel was used as an inducer for this transformation (Scheme

4.30).146

Scheme 4.30. A consecutive three-component one-pot synthesis of 2,5-disubstituted

oxazoles.145

In another approach, the synthesis of the oxazole derivatives possessing unsaturated side

chains through a palladium catalyzed cycloisomerization-allylation reaction of

propargylamide derivatives with allyl carbonates using tricyclohexylphosphane (Cy3P) and

1,3-bis(2,6-diisopropylphenyl)imidazole-2-ylidene (IPr·HCl) ligands has been established

(Scheme 4.31).147 The mechanistic steps have been rationalized to be following the

cyclization of propargylamides through the oxypalladation caused by a π-allyl palladium

complex, generated by Pd(0) and allyl ethyl carbonate, followed by reductive elimination147

unlike previously studied mechanisms where the coupling step was followed by the

acid145/base144 cycloisomerization step.

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Scheme 4.31. Palladium catalyzed cycloisomerization-allylation of propargylamide with allyl

carbonate for 2,5-disubstituted oxazole synthesis.147

4.5.2. Intramolecular Cycloisomerization of Terminal Propargylamides

The capability of palladium to induce cycloisomerization was employed for heterocyclization-

alkoxycarbonylation to synthesize (E)-5-(alkoxycarbonyl)methylene-3-oxazolines (Scheme

4.32).148 The process of heterocyclization has been shown to be strongly dependent upon

the structural features of propargylamides and the nature of the solvent. Moreover, the

oxazolines so formed can further undergo rearrangement to six-membered ring systems

under acidic conditions.

Scheme 4.32. Pd-catalyzed oxidative cyclization-alkoxycarbonylation of propargylamides.148

The concept of Pd(II) catalyzed oxidative cycloisomerization gave rise to the syntheses of 2-

substituted-5-oxazolecarbaldehydes (Scheme 4.33).149 However, the mechanistic role of

palladium was not clear, it was deduced to undergo complexation with triple bond, making it

susceptible to nucleophilic attack (oxygen atom of amide moiety) giving rise to 5-exo-dig

cyclization to the corresponding oxazole structure. The presence of oxidant promoted the

regeneration of active Pd(II) species in catalytic cycle and dehydrogenation of 4,5-

dihydrooxazole-5-carbaldehyde to oxazole.

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Scheme 4.33. Intramolecular Pd(II)-oxidative cyclization of propargylamides to 2-

substituted- 5-oxazolecarbaldehyde.149

Over the years many metal catalytic systems such as silver,150 gold (Au(III)151-153 and

Au(I)),154-155 copper,156 Lewis acid146,157 and non-metal catalyst such as hypervalent iodine

oxidants (PIDA)158 have emerged for cycloisomerization of terminal propargylamides under

relatively milder and robust reaction conditions (Scheme 4.34).

Scheme 4.34. Different metal and non-metal catalytic systems for cycloisomerization of

terminal propargylamides (a,150 b,158 c,157 d,157 e,151-153).

Scheme 4.35, depicts the utility of Cu(I) for the syntheses of a variety of novel 11β-aryl-

17,17-spiro[(4'H,5'-methylene)oxazol]-substituted steroids in moderate to good yields via

cyclization of acylaminoacetylenes.156

Scheme 4.35. Cu(I) catalyzed cycloisomerization of steroidal acylaminoacetylenes to

dihydrooxazole.156

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4.5.3. Intramolecular Cycloisomerization of Non-terminal Propargylamides

PIDA mediated cycloisomerization of non-terminal propargylamides was successful in

furnishing the corresponding oxazole but lower yields of the products were obtained

(Scheme 4.36).158 Under Au(III) reaction conditions, non-terminal propargylamides have

been shown to remain unresponsive to the desired transformation therefore a Au(I) catalyst

was developed that gave rise to the corresponding oxazolines (via 5-exo-dig cyclization) and

oxazines (via 6-endo-dig cyclization) using gold-carbene catalyst system.154 The selectivity

between the two products was strongly dependent upon the nature of the catalyst system

and the alkyl side chain length of the propargylamides. No generality of the scope could be

concluded as mixture of products was obtained using various substituted propargylamide

substrates. However, oxazoline derivatives were further converted to peroxide oxazole

derivatives under oxidizing conditions (atmospheric oxygen) (Scheme 4.36).155

Scheme 4.36. PIDA157 and Au(I)154-155 mediated cycloisomerization of non-terminal

propargylamides.

4.5.4. Coupling and Cycloaddition Reactions of Propargylamides

Syntheses of modified homodimeric "Gonadotropin Releasing Hormone Receptor (GnRHR)"

antagonist dimers with rigid functionalities such as bistriazole with hydrophilic polyethylene

glycol (PEG) spacer159 (Scheme 4.37) and a propargylated aryl system replacing the PEG

spacer,160 have been accessed via cycloaddition and coupling reaction, respectively.

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Scheme 4.37. Synthesis of homo-bistriazole GnRHR ligands via click reaction of

propargylated receptor with bisazide ethyleneglycol.159

Continuation of this study led to the replacement of the bistriazole system with a more rigid

hydrophobic propargylated aryl system that was synthesized by the coupling between

terminal alkyne moiety of propargyl protected amino acid derivatives with the respective

iodoaryl systems (Scheme 4.38).160

Scheme 4.38. Synthesis of homodimeric GnRHR antagonists having a rigid bis

propargylated benzene core.160

While in another study, deoxynucleoside derivatives have been synthesized by coupling of

propargylated trifluoroacetyl systems with iodo substituted pyramidinones, for their biological

evaluation (Scheme 4.39).161

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Scheme 4.39. Synthesis of propargylated trifluoroacetyl derivative of pyramidinone.161

Results and Discussion

51

5. Results and Discussion

5.1. Optimization of Enzyme Catalyzed Aminolysis

5.1.1. Solvent Selection

The solvent selection for the enzyme catalyzed reaction of interest is not straightforward and

there are always exceptions to the general rules. However log P values (logarithm of the

partition coefficient of a given solvent between 1-octanol and water)34 have been well

established as one of the most reliable criteria for solvent selection for an enzyme catalyzed

process.2,10,162,163 Solvents possessing intermediate polarity, that are non-substrate to

enzyme, have been suggested to be the most suitable ones since the highly polar solvents

may render the enzyme inactive by removing the essential water layer off the active site of

the enzyme.164 The solvent selection also crucially depends upon the substrates as the

selection becomes difficult if the substrates exhibit dissimilar solubility characteristics.164

In the present study, different solvents have been screened based on their log P values, for

the intended aminolysis and optimization study was conducted using most commonly used

lipase, CAL-B (Novozyme® 435) (Scheme 5.1). The screened organic solvents with their

respective log P values are mentioned in Table 5.1.

Scheme 5.1. Model reaction for Novozyme® 435 catalyzed aminolysis.

Table 5.1. log P Values of different organic solvents screened for optimization of Novozyme®

435 catalyzed aminolysis (Scheme 5.1).

Entry

Solvents

log P

t [h] until product 3a

detection by GC-MS

1 n-hexane34 3.5 2.5

2 cyclohexane34 3.2 2.5

3 toluene34 2.5 -

4 MTBE165 1.49 0.5

5 DIPE34 1.9 0.5

6 DCM166 2.0 -

7 THF34 0.49 -

Results and Discussion

52

Entry

Solvents

log P

t [h] until product 3a

detection by GC-MS

8 1,4-dioxane34 -1.1 -

9 t-BuOH34 0.80 -

10 MeOH34 -0.76 -

11 acetonitrile34 -0.33 -

12 acetone34 -0.23 -

13 1,2-dimethoxyethane - -

The results can be rationalized on the basis of the polarity of the solvents used. In highly non

polar solvents such as n-hexane and cyclohexane, the product formation was observed after

2.5 h of reaction time and the product 3a so formed, began to crystallize out in the reaction

vial. This observation has been attributed to the poor solubility of the product 3a in these

solvents. The fastest progress of the reaction was observed when ether solvents such as

MTBE and DIPE were used but when the moderately polar to highly polar solvents were

employed (Table 5.1, entries 7-12), no product formation was observed. Therefore, a subtle

balance between the polarity of the solvent employed and its hydrophilicity, has been

considered important to maintain. The solvent to be used has to be polar enough to

solubilize the polar substrates but not hydrophilic to the extent rendering the enzyme

inactive. So, for the present study, the use of nonpolar solvents such as n-hexane and

cyclohexane was not appropriate because these solvents do not offer a wide spectrum of

solubility options for the different types of ester substrates 1 to be used. Since the ether

solvents were the most efficient ones, therefore, MTBE was chosen as solvent of choice.

5.1.2. Screening of Enzymes

According to the literature pertaining to biocatalysis, lipases have been the enzymes of

choice for esterification and transesterification reaction but their utility as a biocatalyst for

aminolysis is rather limited.74 In the past few decades the studies attributed towards

applicability of CAL-B for aminolysis and ammonolysis, have credited CAL-B for such

transformations.85-94,101-104,111-117 Not only CAL-B but also the utility of PCL has been

established in the past years for aminolysis reactions.105-107 CAL-A has been shown to be

active in aminolysis of esters but not in the esterification reactions.101 According to conducted

literature survey, no reference was available stating the aminolysis of an ester substrate

using propargylamine (2). Various lipases such as PPL, CRL, PCL (immobilized on

immobead® 150), CAL-A (immobilized on immobead® 150), CAL-B (two types of

commercially available immobilized CAL-B have been used : a) immobilized on immobead®

Results and Discussion

53

150 and, b) immobilized on acrylic resin that is commercially known as Novozyme® 435) and

protease from Bacillus licheniformis (Alcalase CLEA® ) were used for the model aminolysis

reaction (Scheme 5.1). The results of enzyme screening have been shown in Table 5.2.

Table 5.2. Screening of different enzymes for aminolysis of ester 1a for the synthesis of

propargylamide 3a shown in Scheme 5.1.

Entry

Enzyme

Mode of Immobilization

t [h]

3a

Yield

(%)

1 CAL-B Immobilized on immobead® 150 24 35

2 CAL-B Immobilized on immobead® 150 48 67

3 CAL-B Immobilized on acrylic resin

(Novozyme® 435)

24 68

4 CAL-A Immobilized on immobead® 150 24 -

5 CRL - 24 -

6 PPL - 24 -

7 PCL - 24 -

8 Alcalase CLEA® 24 -

According to the optimization studies mentioned in Table 5.2, only CAL-B (Novozyme® 435)

was able to catalyze the corresponding aminolysis by propargylamine (2) (Table 5.2, entries

1-3) while the rest of the enzymes failed to catalyze this reaction. CAL-B (Novozyme® 435)

exhibited similar results in half the time (Table 5.2, entry 3) that was taken by CAL-B

immobilized on immobead® 150 (Table 5.2, entry 2) therefore Novozyme® 435 was chosen

for the catalysis of aminolysis of the ester 1a. This observation can be rationalized by taking

into account the mode of immobilization of CAL-B on solid support. In case of Novozyme®

435, CAL-B is immobilized on hydrophilic anionic resin, Lewatit® E, through ionic binding101

while in case of immobead® 150, the enzyme is immobilized on cross linked co-polymer of

methacrylate (carrying oxirane groups) through covalent binding. Some loss of activity has

always been reported in case when the enzyme is bound by covalent binding and

consequently the residual activity of the enzyme is not believed to exceed 60-80 % while

such an extent of loss of activity has not been observed when the mode of binding is ionic.34

This explains the longer time requirement of CAL-B immobilized on immobead® 150 for

furnishing the yield of the corresponding product 3a, that was produced by Novozyme® 435

Results and Discussion

54

in half the time. Effect of the mol ratio of ester 1 to propargylamine (2) and the amount of

enzyme used, on the product 3a yield was determined. The results are shown in Table 5.3.

Table 5.3. Effect of mol ratio of ester 1a and propargylamine (2) on isolated yield of the

product 3a at 45 °C and 24 h of reaction time (Scheme 5.1).

Entry

Amount of CAL-B

(Novozyme® 435)

(% w/w of ester substrate 1)

Mol Ratio

Ester 1a:Propargylamine (2)

3a

Yield

(%)

1 10 % 1:1 13

2 10 % 1.2:1 14

3 20 % 1:1 14

4 20 % 1.2:1 16

5 30 % 1.2:1 39

6 40 % 1.2:1 44

7 50 % 1:1 60

8 50 % 1.2:1 68

9 50 % 1:1.2 56

10 50 % 2:1 67

11 50 % 1:2 53

12 100 % 1.2:1 62

13 100 % 2:1 59

14 100 % 1:2 51

The data mentioned in Table 5.3, showed that the highest yield of the product 3a was

obtained using a slight excess of ester 1a with Novozyme® 435 in a 50% w/w ratio to ester

1a (Table 5.3, entry 8). Therefore, these reaction conditions were chosen for further

optimization of Novozyme® 435 catalyzed aminolysis of esters 1 by propargylamine (2).

5.1.3. Substrate (Acyl Donor) Selection

Studies pertaining to structural features of CAL-B established that, compared to other

lipases, it possesses rather limited available space in the active site pocket. This feature is

believed to be responsible for its enhanced substrate specificity.167 X-ray crystallographic

studies revealed the active site to be composed of a relatively more spacious acyl channel

than the alcohol channel168 and based on this, CAL-B is expected to exhibit comparatively a

broad specificity for acyl donors.169

Results and Discussion

55

A wide range of acyl donors (ester substrates) have been successful in establishing their

suitability for CAL-B catalyzed transesterification reaction.167 This observation prompted the

screening of different ester substrates for the synthesis of structurally diverse

propargylamides 3 through aminolysis that, in turn, would bring the structural variety to the

corresponding products intended to be obtained by one-pot processes.

5.1.3.1. Substrates with Heteroatom Side Chain Linkers

There exists a correlation between structural features of ester substrates 1 and the operating

temperature dependency of CAL-B (Novozyme® 435) catalysis. The Novozyme® 435

catalyzed aminolysis of ester substrate 1a employed in the model reaction (Scheme 5.1)

could produce the corresponding amide product 3a only when the temperature of 40-45 °C

was maintained and yield did not exceed 68 % despite of longer reaction time and increased

catalyst loading (Table 5.3, entries 12 and 13). So, the next step was to search for the ester

substrates 1 compatible with Novozyme® 435. Enhanced reactivity of ester substrates with

the oxygenated side chain linkers in PCL catalyzed aminolysis has been reported in

literature.105-107 In the present work not only the effect of presence of oxygen but also the

other heteroatoms, such as sulphur and nitrogen, in the side chain of the ester has been

studied and an enhanced acceptability of such ester substrates has been observed. Table

5.4, shows the operating temperature dependency of Novozyme® 435 catalyzed aminolysis

on the nature of ester substrates 1 used.

Scheme 5.2. Novozyme® 435 catalyzed aminolysis using different ester substrates 1.

Results and Discussion

56

Table 5.4. Effect of structural features of the ester substrates 1 on the operating temperature

and time taken by Novozyme® 435 catalyzed aminolysis (Scheme 5.2).

Entry

Ester Substrates 1

T [°C]

t [h]

3

Yield (%)

1

1a

RT 24 -

45 24 68

2

1b

RT 24 -

45 24 62

3

1c

RT 24 -

45 24 73

4

1d

RT-45

24

-

5

1e

RT

6

86

6

1f

RT

6

86

7

1g

RT

4

95

Results and Discussion

57

Entry

Ester Substrates 1

T [°C]

t [h]

3

Yield (%)

8

1h

RT

8

74

9

1i

RT

8

76

10

1j

RT

8

82

11

1k

RT

4

80

12

1l

RT-45

-

-

The results, shown in Table 5.4, indicate a clear demarcation between various types of

esters 1 used. The activated ones are those containing hetero atom linkers in their side

chain (Table 5.4, entries 5-11) and consequently exhibited higher acceptability by the

enzyme resulting high product 3 yield in shorter period of time at room temperature. While

the rest produced satisfactory yields of the respective products 3, relative to activated esters

Table 5.4, entries 5-11), over a prolonged reaction time as well as operating temperature of

45 °C. The high acceptability of the activated ester substrates 1e-k can be rationalized

based on the inductive effect of the hetero atom side chain linkers in their structure. The

presence of the hetero atom in the side chain renders the carbonyl moiety of the ester more

electrophilic, hence making it more susceptible for the enzyme attack. While such a

possibility does not exist in the case of esters without hetero atom in their side chains and

Results and Discussion

58

hence results in relatively a low acceptability of these substrates by the enzyme. Site

selective preference of Novozyme® 435 of such sort was further exploited when diester 1k

(Table 5.4, entry 11) was used and the reaction selectively took place at the ester moiety

with oxygenated side chain linker. However the reaction did not proceed beyond the mono

amidation product mainly due to the insolubility of the product 3k in MTBE. Methylbenzoate

(1d), (Table 5.4, entry 4) could not produce the corresponding "aminolyzed" product but

ester substrate 1c (Table 5.4, entry 4) furnished the respective amide product, though the

substrates 1c and 1d differ only by the presence of a methylene group between aromatic

ring and ester linkage thus showcasing the dependence of selectivity of enzyme upon subtle

structural differences.

5.1.3.2. N-Protected Amino Acids as Ester Substrates

Since Novozyme® 435 is able to successfully catalyze the intended aminolysis reaction

(Scheme 5.1), therefore it was considered interesting to assess its receptive ability for

methyl esters of N-protected amino acids for aminolysis by propargylamine (2). Lipases,

unlike proteases, do not possess amidase activity75 and therefore it is always interesting to

employ lipases as the catalyst of choice for peptide synthesis. PPL is considered to be the

only class of lipases that is active towards peptide synthesis while CRL catalyzed reactions

involving the use of N-protected amino esters as electrophiles and free amino acids as

nucleophiles.170 Subtilisin has been studied to catalyze the incorporation of D-amino acids

(via acylation) into the peptide sequence.171 Synthesis of β-dipeptides has been reported

using CAL-A for catalyzing the acylation of β-amino esters.114 Since Novozyme® 435 has not

previously been employed for acylation reactions involving amino acids, therefore, in present

study methyl esters of different N-protected amino acids 1m-r, were used as ester

substrates and the effect of different protecting groups towards the reactivity of amino acids

was determined. The results revealed that CAL-A and CRL could not catalyze the intended

reaction (Scheme 5.3) but Bacillus licheniformis protease (Alcalase CLEA®) could produce

the desired respective, 3n and 3o, product over prolonged reaction time (24-48 h) in lower

yield (15-20 %).

Scheme 5.3. Novozyme® 435 catalyzed aminolysis of methyl ester of N-protected amino

acids 1m-r.

Results and Discussion

59

Results (Table 5.5) suggested that the catalytic activity of Novozyme® 435 was highly

dependent upon the nature of the protecting group employed. Different N-protected D- and

L-amino esters were used and the results showed that amino esters protected with a Boc

group were not suitable as no product formation was observed. This can be attributed to the

steric bulk of the Boc group making it unbefitting for Novozyme® 435. While N-protected D-

alanine methyl esters, 1n and 1o (Table 5.5 entry 1), exhibited more flexible behaviour

compared to its counterpart N-protected L-alanine methyl ester 1p, as D-alanine methyl

ester derivatives, protected by TFA 1n and Cbz 1o, were active towards aminolysis but L-

alanine methyl ester could only be converted to the corresponding amide when protected by

Cbz group (Table 5.5, entry 2), in shorter time period. This observation also suggested that

selectivity can be achieved in instances involving both D- and L-amino acids owing to the

differences of behaviour these N-protected amino methyl esters exhibited towards

Novozyme® 435 catalyzed aminolysis when protected by different protecting groups.

Table 5.5. Effect of the nature of different protecting groups on the receptive behaviour of

Novozyme® 435 towards different methyl esters of N-protected amino acids.

Entry Ester Substrate 1 t [h] Propargylamide 3

1

1m : PG = Boc

1n : PG = TFA

1o : PG = Cbz

4

3m (no product formation)

3n (62 %)

3o (82 %)

2

1p

4

3p (80 %)

3

1q

24

3q (68 %)

4

1r

24

3r (53 %)

Results and Discussion

60

5.1.3.3. Ester Substrates with Heterocyclic Systems

Ester substrates with heterocyclic systems, have also been screened for their susceptibility

to aminolysis catalyzed by Novozyme® 435. The results are shown in Table 5.6. The ester

substrates with furan, thiophene and N-substituted piperidine systems (Table 5.6, entries 1-

3) were successfully converted to the corresponding products 3 while pyrrole, both

unsubstituted 1v and N-methyl substituted 1w (Table 5.6, entries 4 and 5), pyridine 1x,

(Table 5.6, entry 6) and benzothiophene system 1y (Table 5.6, entry 7), failed to furnish the

corresponding amide products.

Table 5.6. Ester substrates 1 with heterocyclic systems for Novozyme® 435 catalyzed

aminolysis.

Entry

Ester Substrates 1

T [°C]

t [h]

3

Yield (%)

1

1s

RT 24 -

45 24 71

2

1t

RT 24 -

45 24 77

3

1u

RT 24 -

45 24 70

4

1v

RT 24 -

45 24 -

5

1w

RT 24 -

45 24 -

Results and Discussion

61

Entry

Ester Substrates 1

T [°C]

t [h]

3

Yield (%)

6

1x

RT 24 -

45 24 -

7

1y

RT 24 -

45 24 -

5.1.3.4. Methyl propiolates as Acyl Donor

Ester substrate with multiple bonds in their side chain 1z and 1aa were screened for their

suitability for aminolysis. Novozyme® 435 catalyzed aminolysis of methyl propiolate (1z) with

propargylamine (2) not only gave rise to the corresponding desired amide product 3z but

also the formation of the Michael adduct 3za was observed in GC-MS analysis, an

experimental observation contrary to the previously reported selective amidation (1,2-

addition) by CAL-B in MTBE96,97 (Scheme 5.4).

Scheme 5.4. Reaction outcome of aminolysis of methyl propiolate (1z) by propargylamine

(2).

Since the aminolysis of methyl propiolate (1z) resulted in the formation of both amide (1,2

addition) and undesired Michael (1,4-addition) adduct product, therefore, in order to prevent

the formation of the Michael product, a substituted propiolate, such as methyl

phenylpropiolate (1aa), was employed and as expected, no Michael adduct formation was

observed and corresponding amide 3aa was the sole product of Novozyme® 435 catalyzed

aminolysis (Scheme 5.5).

Results and Discussion

62

Scheme 5.5. Aminolysis of methyl phenylpropiolate (1aa) by propargylamine (2).

5.1.3.5. Long Chain Fatty acids

Since long chain fatty acids are natural substrates for lipases, therefore, Novozyme® 435

catalyzed aminolysis by propargylamine (2) of long esters and acids was screened. For this

purpose oleic acid (1bb) and octanoic acid (1cc) were screened. The reaction was allowed

to run at room temperature and when no product formation was observed, the temperature

was increased to 45 °C. The reaction was monitored via both TLC and GC-MS techniques

that showed no product formation in the reaction mixture (Schemes 5.6 and 5.7).

Scheme 5.6. Aminolysis of oleic acid (1bb) with propargylamine (2) catalyzed by

Novozyme® 435.

Scheme 5.7. Aminolysis of octanoic acid (1cc) with propargylamine (2) catalyzed by

Novozyme® 435.

Thus the screening of various possible ester substrates 1 for the intended lipase catalyzed

aminolysis revealed the suitability and compatibility of Novozyme® 435 with a number of

possible ester substrates 1 under relatively mild reaction conditions.

Results and Discussion

63

5.2. Optimized CAL-B (Novozyme® 435) Catalyzed Aminolysis

With the exception of activated esters, 1e-k, the rest required the reaction temperature of

45 °C for furnishing the corresponding propargylamides 3 in satisfactory yields. Therefore

the optimum temperature of Novozyme® 435 catalyzed aminolysis was chosen to be 45 °C.

Depending upon the type of ester substrates 1 employed, the reaction was completed

between 4 to 24 h of reaction time (Scheme 5.8).

Scheme 5.8. Optimized Novozyme® 435 aminolysis of various ester substrates 1 by

propargylamine (2).

In order to develop the efficacy of the optimized Novozyme® 435 catalyzed aminolysis, a

comparison was made between the yields of the products 3 obtained by base (triethylamine)

catalyzed (synthesized and literature reported values) and Novozyme® 435 catalyzed

aminolysis as shown in Table 5.7. The comparison revealed that comparable yields of the

corresponding propargylamides 3 could be obtained by Novozyme® 435 catalyzed protocol

under milder reaction conditions.

Table 5.7. Comparison of yields of propargylamides 3 prepared by base (NEt3) catalyzed

and Novozyme® 435 catalyzed aminolysis.

Entry

Propargylamides

3

Base (NEt3)

catalyzed

aminolysis

Yield (%)

Novozyme® 435

catalyzed

aminolysis

Yield (%)

1

3a

73a

68b

2

3b

75a

62b

Results and Discussion

64

Entry

Propargylamides

3

Base (NEt3)

catalyzed

aminolysis

Yield (%)

Novozyme® 435

catalyzed

aminolysis

Yield (%)

3

3c

92c

73b

4

3j

84d

82e

5

3s

85f

71b

6

3t

65g

77b

Reaction conditions: aNEt3 (1.0 eq), DCM, 0-20 °C, 4 h,

b24 h,

cliterature reported value,

172 dliterature

reported value,173 e

4 h, fliterature reported value,

151 gliterature reported value.

149

5.3. Optimization Studies for Chemoenzymatic One-pot Synthesis of 1,4-Disubstituted

1,2,3-Triazoles 5

Classical reaction conditions for the Cu(I) catalyzed click reaction involves the application of

t-BuOH:H2O (1:1) as solvent of choice and the reaction usually takes place at room

temperature. The application of such sort of trivial reaction conditions was not possible in the

present case as most of the propargylamides 3, were not soluble in t-BuOH. Therefore, other

reported reaction conditions involving the use of solvents other than t-BuOH for click reaction

were employed. Propargylamides 3 have been established as substrates that are known for

their low reactivity towards Huisgen 1,3-dipolar cycloaddition with azides,174 but have been

reported to exhibit high reactivity in Cu(I) catalyzed azide-alkyne cycloaddition (CuACC) and

produced 1,2,3-triazoles in quantitative yields when DCM was used as solvent.175 Table 5.8,

summarizes the outcome of the optimization study of one-pot click reaction using different

solvents, copper catalyst species and ligands.

Results and Discussion

65

Scheme 5.9. Chemoenzymatic one-pot synthetic scheme of 1,4-disubstituted 1,2,3-triazole

5e.

Of all the reaction conditions employed, only the ones involving the use of base and Cu(I)

stabilizing ligand, could produce the corresponding 1,2,3-triazole 5e in satisfactory yield

(Table 5.8, entries 9-11). The possible interaction(s) of the enzyme with the respective metal

catalytic species leading to its consequent reduction in concentration in the same reaction

mixture, has been a topic that is not being addressed vividly. But the success of ligand

promoted click reaction in a one-pot methodology, pointed towards the diminishing

concentration of Cu(I) species in the reaction mixture when conducted under ligand free

conditions. Recently, a relatively new concept for CuACC has been introduced involving the

application of Cu2O as direct Cu(I) source in the presence of carboxylic acids as bidentate

Cu(I) stabilizing ligands176a-c and the incorporation of this concept has been successful in a

one-pot fashion (Table 5.8, entry 11).

Results and Discussion

66

Table. 5.8. Optimization studies of chemoenzymatic one-pot click reaction (Scheme 5.9).

Entry

Cu Source

Reducing

agent

(Na-ascorbate)

Base

Ligand

Solvent

T

[°C]

t

[h]

5e

Yield

(%)

1 CuSO4·5H2O

(5 mol%)

20 mol% - - THF:H2O

(2:1)

RT 48 35

2 CuSO4·5H2O

(5 mol%)

20 mol% - - EtOH:H2O

(1:1)

RT 24 52

3 CuSO4·5H2O

(4 mol%)

20 mol% Na2CO3

(20 mol %)

- EtOH:H2O

(1:1)

RT 24 42

4 CuSO4·5H2O

(4 mol%)

20 mol% Na2CO3

(20 mol %)

- EtOH:H2O

(2:1)

RT 24 38

5

Cu(OAc)2·H2O

(6 mol%)177

6 mol%

- -

MeCN:

H2O (1:1)

RT 42 72

Na2CO3

(20 mol %)

L-Proline

(20 mol%)

RT 24 61

Na2CO3

(20 mol %)

L-Proline

(20 mol%)

45 19 85

6

CuSO4·5H2O

(5 mol%)175

15 mol%

-

-

DCM:

H2O

(1:1)

RT 48 60

45 24 63

7

CuI (10 mol%)

-

DIPEA

(1.0 eq)

Ethylene

diamine

(1.0 eq)

Dry THF

under

argon

RT

24

61

8 CuSO4·5H2O

(5 mol%)178

20 mol% Na2CO3

(20 mol %)

L-Proline

(20 mol%)

DMSO:H2O

(9:1)

45 16 53

9

CuSO4·5H2O

(5 mol%)

20 mol%

Na2CO3

(20 mol %)

L-Proline

(20 mol%)

MeOH:

H2O (1:1)

RT 24 61

45 19 85

10

CuSO4·5H2O

(5 mol%)

20 mol%

Na2CO3

(20 mol %)

L-Proline

(20 mol%)

DCM:

H2O (1:1)

45

19

82

11

Cu2O

(4 mol%)176

-

-

PhCO2H

(8 mol %)

H2O:MeOH

RT 8 61

45 4 83

Results and Discussion

67

Considering the outcome of the one-pot triazole synthesis under different reaction

conditions, entries 9 and 10 in Table 5.8, were two of the three high yielding sets of reaction

conditions for triazole 5e synthesis but these reaction conditions were disadvantageous as

these involved the usage of additives and prolonged reaction time compared to carboxylic

acid promoted CuACC sequence (Table 5.8, entry 11). Table 5.9, shows the comparison of

the isolated yields of the products obtained by using both the optimized reaction conditions.

Table 5.9. Yield of the 1,2,3-triazoles 5 obtained by chemoenzymatic one-pot synthesis.

Entry

1,2,3-Triazoles

Yield (%)

Catalyst System A

Catalyst System B

1

5a

71

73

2

5b

61

63

3

5e

83

85

4

5f

70

72

5

5h

74

51

Catalyst System A: Cu2O (4 mol%), PhCO2H (8 mol%), 45 °C, 4 h, MeOH:H2O (1:1). Catalyst System

B: CuSO4·5H2O (5 mol%), Na-ascorbate (20 mol%), L-Proline (20 mol%), Na2CO3 (20 mol%) 45 °C,

18-20 h, MeOH:H2O (1:1). MeOH as co solvent was used only when propargylamide 3 was insoluble

in MTBE.

Results and Discussion

68

Since, more or less, the same yield of the respective 1,2,3-triazole products 5 was obtained

using the two sets of reaction conditions mentioned in Table 5.9, therefore, the reaction

conditions involving the use of less additives and a shorter period of reaction time i.e. Cu2O

as direct Cu(I) source in the presence of benzoic acid as bidentate ligand, was chosen for

the one-pot sequence. Various commercially available azides 4a-e were employed, but only

azides 4a and 4b could successfully participate and generate the corresponding triazoles 5

(Scheme 5.10).

Scheme 5.10. Screening of azides 4a-e for the synthesis of corresponding triazole systems

5 via chemoenzymatic one-pot procedure.

Table 5.10. Azides 4a-e used in chemoenzymatic one-pot sequence for the synthesis of

corresponding triazoles 5.

Entry

Azides

Yield (%)

5

1

4a

5b (61 %)

2

4b

5ff (63 %)

3

4c

-

4

4d

-

Results and Discussion

69

Entry

Azides

Yield (%)

5

5

4e

-

5.4. Synthesis of Triazoles 5 via In situ Azide Generation

In order to circumvent the use of organic azides, considering their perilous nature, the

concept of the synthesis of 1,2,3-triazole system 5 via in situ generation of benzyl azide (4a)

has also been carried out but no satisfactory corresponding product 5a yield was obtained.

The optimization studies were conducted in a one-pot fashion and also utilizing isolated

propargylamide 3a but no adequate outcome was obtained (Scheme 5.11).

Scheme 5.11. Synthesis of 1,2,3-triazole 5a via in situ generation of benzyl azide (4a).

Table 5.11. Chemoenzymatic one-pot procedure via in situ benzyl azide (4a) generation.

Entry Catalyst System T [°C] Solvent 5a Yield (%)

1

Na-ascorbate (20 mol%)

L-Proline (20 mol%)

Na2CO3(20 mol%)

CuSO4·5H2O (5 mol%)

45-55

DCM

36

2

Na-ascorbate (20 mol%)

L-Proline (20 mol%)

Na2CO3(20 mol%)

CuSO4·5H2O (5 mol%)

45-55

MeOH

44

Results and Discussion

70

Entry Catalyst System T [°C] Solvent 5a Yield (%)

3 Cu2O (4 mol%)

Benzoic acid (8 mol%)

45-55 MeOH 43

4 aCu2O (4 mol%)

Benzoic acid (8 mol%)

45-55 MeOH 45a

aReaction was commenced using isolated propargylamide 3a.

5.5. Optimized Chemoenzymatic One-pot Synthesis of Amide Ligated 1,4-

Disubstituted 1,2,3-Triazoles 5

The screening of different copper sources as possible Cu(I) catalytic species, various

solvents, bases and ligand stabilizing species, led to the optimized chemoenzymatic one-pot

synthesis of amide ligated 1,4-disubstituted 1,2,3-triazoles 5 (Scheme 5.12). The optimized

one-pot methodology exhibited a fair level of compatibility with a wide variety of acyl donors

1 except for the ester substrates with functionalities that might act as Cu(I) stabilizing motifs

(1g, 1q, 1r, 1u) that in turn failed to produce the corresponding triazole products.

Scheme 5.12. Optimized chemoenzymatic one-pot synthesis of 1,4-disubstituted 1,2,3-

triazoles 5.

Results and Discussion

71

Table 5.12. Triazoles 5 obtained by chemoenzymatic one-pot sequence using benzyl azide

(4a).

Entry

Product 5

Yield

(%)

Entry

Product 5

Yield

(%)

1

5a

71

7

5j

66

2

5b

61

8

5k

51

3

5e

83

9

5n

59

4

5f

70

10

5o

(using substrate 1o)

70

5

5h

85

11

5p

(using substrate 1p)

73

6

5i

74

12

5s

68

Results and Discussion

72

Entry

Product 5

Yield

(%)

Entry

Product 5

Yield

(%)

13

5t

35

15

5dd

40

14

5aa

71

16

5ee

61

Table 5.13. Triazoles 5 obtained by chemoenzymatic one-pot sequence using azidomethyl

phenyl sulphide (4b).

Entry

Product 5

Yield

(%)

Entry

Product 5

Yield

(%)

17

5ff

63

19

5hh

59

18

5gg

78

20

5ii

85

Results and Discussion

73

5.6. Intended Modification of Amidation-Coupling-Cycloisomerization (ACCI)

Sequence

Propargylamides 3 have been used as an entry to multicomponent one-pot synthesis of

oxazoles 7 via amidation-coupling-cycloisomerization (ACCI) sequence.145 In this context, a

modification of this sequence has been carried out by replacing base (NEt3) catalyzed

aminolysis of benzoyl chloride (4g) (Scheme 5.13) with Novozyme® 435 catalyzed

aminolysis. Base catalyzed aminolysis does not allow the liberty to introduce functional

group diversity in the corresponding product due to the possibility of their sensitivity towards

the base. However base could be replaced by such catalytic system that would allow the

introduction of base labile groups and present a relatively mild approach, therefore it was

considered worthwhile to incorporate the concept of Novozyme® 435 catalyzed aminolysis to

the designed synthesis of oxazoles 7 (Scheme 5.13).

Scheme 5.13. Intended chemoenzymatic one-pot synthesis of 2,5-disubstituted oxazoles 7

via amidation-coupling-cycloisomerization (ACCI) sequence.

5.6.1. Optimization Studies of Sonogashira Coupling Reaction

Optimization studies were conducted by carrying out one-pot synthesis using the reaction

conditions for coupling reported previously (Scheme 5.14).145

Results and Discussion

74

Scheme 5.14. First attempt to chemoenzymatic one-pot synthesis of coupled products 6a

and 6b.

The expected coupled products 6a and 6b could not be obtained and the GC-MS spectrum

of the reaction mixture revealed the presence of unreacted starting material 1 while almost

complete consumption of benzoyl chloride (4g) but no corresponding product 6 formation

was observed in the spectrum. Moreover there was a prominent peak of m/z = 136 in GC-

MS spectrum corresponding to the molecular mass of methyl benzoate. The formation of

methyl benzoate can be rationalized since methanol has been formed as the by product of

the Novozyme® 435 catalyzed aminolysis, so it immediately reacted with benzoyl chloride

(4g) forming methyl benzoate which resulted in the low concentration of benzoyl chloride in

the reaction mixture to be able to couple with propargylamide 3. So in order to overcome

this, reaction was repeated using 5 Å molecular sieves for quenching the methanol formed

during aminolysis. The GC-MS profile of the reaction mixture showed the presence of

unreacted species, 3a, 3b or 4g, but no product 6 formation was observed. In another

attempt, the sequence was performed in stepwise manner by reacting propargylamide (3a

and 3b, isolated separately) with benzoyl chloride (4g) using standard conditions for

Sonogashira coupling145 but no product formation, 6a or 6b, was observed. This indicated

the inability of the chemical catalytic system (Pd(II)/Cu(I)) in catalyzing the desired reaction.

Therefore the coupling step, using isolated propargylamide 3, was optimized by employing

different catalytic systems. The results of initial optimization studies are shown in Table 5.14.

Results and Discussion

75

Table 5.14. Optimization studies of coupling reaction, shown in Scheme 5.14, using

standard conditions for Sonogashira coupling reaction.145

Entry Solvent T [°C] Other Additives t

[h]

Yield (%)

1 THF RT-50 - 1 -

2 THF RT-50 5 Å molecular sieves 20 -

3 1,4-dioxane RT-70 5 Å molecular sieves 20 -

Reaction conditions for coupling step: PdCl2(PPh3)2 (2 mol%), CuI (4 mol%), NEt3 (1.0 eq), benzoyl

chloride (4g, 1.0 eq).

Since this strategy possessed the inherent problem of the presence of methanol in the

reaction mixture, therefore the attention was diverted towards using starting materials that

are not reactive towards methanol and would successfully get engaged into the coupling

reaction. So for this purpose, iodobenzene (4h) was used as a coupling partner as shown in

Scheme 5.15, and the results of further optimization studies, only focusing on Sonogashira

coupling, are shown in Table 5.15.

Scheme 5.15. Optimization studies of Sonogashira coupling reaction using iodobenzene

(4h) with the possibility of the formation of cycloisomerization to oxazole systems 6ca or 7a.

Results and Discussion

76

Table 5.15. Results of optimization studies of Sonogashira reaction shown in Scheme 5.15.

Entry Catalyst

System

Ligand Solvent T

[°C]

Base/Additive Yield

(%)

1 aPd(II), Cu(I) - THF RT-50 NEt3 (1.0 eq) -

2 aPd(II), Cu(I) - 1,4-dioxane RT-70 NEt3 (1.0 eq) 13

3 aPd(II), Cu(I) - 1,4-dioxane RT-70 Pyrrolidine (1.0 eq) 25

4 aPd(II), Cu(I) - 1,4-dioxane RT-70 Pyrrolidine (1.5 eq) 33

5 bPd(0), Cu(I) - DMF 45 Pyrrolidine:DMF

(1:4 v/v)

75

6 cPd(0) P(2-furyl)3 MeCN 45 NaOt-Bu (2.0 eq) -

7 dPd(0) IPr·HCl MeCN 45 NaOt-Bu (2.0 eq) -

8 ePd(II) IPr·HCl MeCN 45 K2CO3/TBAC -

9 fPd(0), Cu(I) - DMF RT NEt3 (1.0 eq) 53

10 gPd(0), Cu(I) - DMF 45 DIPEA (1.5 eq) 67

11 hPd(0), Cu(I) - DMF 45 Pyrrolidine (1.0 eq) 56

12 iPd(0), Cu(I) - DMF 45 Pyrrolidine (1.5 eq) 85

13 jPd(0), Cu(I) - DMF 45 DABCO (1.0 eq) 57

14 kPd(0), Cu(I) - DMF 45 DBU (1.0 eq) 69

15 lPd(0), Cu(I) - DMF 45 TMG (1.0 eq) 83

Reaction Conditions: aPdCl2(PPh3)2 (2 mol%), CuI (4 mol%), 24 h,

145 bPd(PPh3)4 (5 mol%),

CuI (1 mol%), 6 h,160 c

Pd2(dba)3 (2.5 mol%), P(2-furyl)3 (10 mol%), 24 h,144 d

Pd2(dba)3 (2.5 mol%),

IPr·HCl (10 mol%), 24 h,147 e

Pd(OAc)2 (5 mol%), IPr·HCl (10 mol%), K2CO3 (2.0 eq), TBAC (2.0 eq),

24 h, fPd(PPh3)4 (2 mol%), CuI (4 mol%), 8-24 h,

gPd(PPh3)4 (2 mol%), CuI (4 mol%), 6 h,

hPd(PPh3)4

(2 mol%), CuI (4 mol%), 6-8 h, iPd(PPh3)4 (2 mol%), CuI (4 mol%), 4 h,

jPd(PPh3)4 (2 mol%), CuI (4

mol%), 6-8 h, kPd(PPh3)4 (2 mol%), CuI (4 mol%), 6-8 h,

lPd(PPh3)4 (2 mol%), CuI (4 mol%), 1-2 h.

Electron deficient ligands are considered favorable for promoting the coupling reaction while

strongly coordinating ligand (electron rich) are considered to retard the coordination of

catalytic palladium species with alkyne for σ-alkenylpalladium complex formation (Scheme

5.16b), hence promoting the intramolecular cycloisomerization to 6ca144 but the implication of

such reaction condition (Table 5.15, entry 6) remained futile in furnishing the corresponding

coupled product 6c and no formation of the intramolecular cycloisomerized product 6ca was

observed. The use of Pd(II) catalyst in conjunction with carbene ligands, has been

established as an efficient approach for Sonogashira coupling of unactivated systems179 and

has also been shown to mediate the cyclization to heterocyclic systems.147,180 But these

reaction conditions were not successful for the coupling reaction either (Table 5.15, entries 7

and 8). The outcome of optimization studies showed Pd(PPh3)4/CuI (Table 5.15, entry 15) to

Results and Discussion

77

be the active and efficient catalyst system for the coupling of propargylamide 3a with

iodobenzene (4h). Tetramethylguanidine (TMG) was the only base, of all the screened

bases, that was successful in furnishing the desired product 6a in good yield by the usage of

1.0 equivalent and under this set of reaction conditions, no cycloisomerized products, 6ca or

7a, were formed. Therefore, these reaction conditions (Table 5.15, entry 15) were chosen for

the one-pot synthesis of corresponding coupling product 6c.

5.6.2. Cycloisomerization of Propargylamides 3

5.6.2.1. Lewis/Protic Acids mediated Cycloisomerization

The third and final step in the sequence shown in Scheme 5.13, is the cycloisomerization of

coupled product 6c to the corresponding 2,5-disubstituted oxazole 7a. Scheme 5.16,

represents an overview of the proposed mechanisms of cycloisomerization of

propargylamides 3 to corresponding oxazole system 7 via complexation of inducers (PIDA,

Pd(II), Lewis acid) with alkyne moiety of propargylamide 6a.

Scheme 5.16. Proposed mechanisms for a) activation of the alkyne moiety by hypervalent

iodine species (PIDA)158 b) σ-alkenylpalladium complex formation149 c) complexation with

Lewis acid.151-153

Propargylamides 3 have been studied to undergo cycloisomerization induced by PTSA at

elevated temperature.145,181 However this set of reaction conditions could not produce the

desired oxazole product 7a (Table 5.16, entry 1). Various reaction conditions involving the

use of Lewis acids and protic acids, were employed but all of them failed to induce the

Results and Discussion

78

cycloisomerization to give the desired product 7a (Scheme 5.17). The optimization studies

for cycloisomerization reaction are mentioned in Table 5.16.

Scheme 5.17. Lewis/protic acid induces cycloisomerization to 2,5-disubstituted oxazole 7a.

Table 5.16. Optimization study for the cycloisomerization reaction shown in Scheme 5.17.

Entry Lewis/Protic Acid Solvent T [°C] t [h] Yield (%)

1 aPTSA THF:t-BuOH (1:1) 60 24 -

2 bMethanesulfonic acid DCM RT-45 24 -

3 cTrifluoromethanesulfonic

acid

DCM 45 24 -

4 dZnCl2 DCM RT-45 24 -

5 eZn(OTf)2 DCM RT-45 24 -

6 fFeCl3 DCM 45 24 -

7 fFeCl3 DCE 60 24 -

8 gAlCl3 DCM 45 24 -

9 hSiO2 DCM RT 24 -

10 iAg(OTf)2 DCM RT-45 24 -

11 jAuCl3 DCM 45 24 -

12 jAuCl3 MeCN 45 24 -

Reaction Conditions for 1.0 eq of isolated 6c: aPTSA (1.0 eq),

145

bMethanesulfonic acid (1.0 eq),

ctrifluoromethanesulfonic acid (1.0 eq),

d0.1M solution of ZnCl2 in diethylether,

157 eZn(OTf)2 (10 mol%),

fFeCl3 (0.5 eq),

157 gAlCl3 (10 mol%),

hSiO2 (300 % w/w),

146 iAg(OTf)2 (10 mol%),

jAuCl3 (10 mol%).

151-

152

5.6.2.2. Oxidative Cycloisomerization

Propargylamides with terminal nonsubstituted alkyne moiety 3 have been reported to

undergo cycloisomerization under oxidative conditions using hypervalent iodine reagents

such as PIDA158 or Pd(II) species with oxidant such as 1,4-benzoquinone.149 Since the use of

Lewis and protic acid could not catalyze the desired transformation (Table 5.16), therefore,

the concept of oxidative cycloisomerization using PIDA and Pd(II) species in the presence of

an oxidizing reagent, was employed but this approach also failed to carry out the required

Results and Discussion

79

transformation (Scheme 5.18). Table 5.17, summarizes the reaction conditions that were

employed using oxidative cycloisomerization concept.

Scheme 5.18. Oxidative-cycloisomerization for oxazole 7a synthesis.

Table 5.17. Optimization studies for oxidative cycloisomerization reaction shown in Scheme

5.18.

Entry Additives for Oxidative

Cycloisomerization

T [°C] 7a

Yield (%)

1 a1,4-Benzoquinone RT-80 -

2 bPd(MeCN)2Cl2, 1,4-BQ RT-80 -

3 cPdCl2, 1N HCl, TBAC RT -

4 dPIDA, AcOH 90 -

Reaction Conditions: a1,4-BQ (1.0 eq), 24 h,

bPdCl2(MeCN)2 (5 mol%), 1,4-BQ (1.0 eq), 24 h,

149

cPdCl2 (5 mol %) TBAC (1.0 eq), 24 h,

dPIDA (1.5 eq), AcOH (5.0 eq), 24 h.

158

Based on the observations pertaining to cycloisomerization studies, it could be concluded

that arylation of propargylamide 3a furnished a rigid propargylated aryl system 6c160 that is

not prone to undergo intended cycloisomerization via acid/base catalysis or σ-alkenyl-

palladium complex formation by Pd(II) species or by the activation of alkyne moiety by

hypervalent iodine (PIDA) (Scheme 5.16).

5.7. Optimized Chemoenzymatic One-pot Amidation-Sonogashira Coupling Sequence

Cycloisomerization could not be induced to generate the desired product by employing the

already reported reaction conditions.149-158 Therefore, the sequence was closed after the

coupling step and eleven coupled products 6 were synthesized using the optimized one-pot

amidation-coupling sequence (Scheme 5.19). However, diiodoaryl systems (Table 5.18,

entries 12 and 13) and iodopyridine (Table 5.18, entry 14) failed to furnish the corresponding

coupled products 6n and 6o.

Results and Discussion

80

Scheme 5.19. Optimized chemoenzymatic one-pot amidation-coupling sequence.

Table 5.18. Arylated propargylamides 6 obtained via amidation-coupling sequence (Scheme

5.19).

Entry Ester Used 1 Iodoaryl

4h-o

Product 6

Yield (%)

1

1a

4h

6c (58 %)

2

1a

4i

6d (62 %)

3

1b

4h

6e (54 %)

4

1c

4h

6f (53 %)

Results and Discussion

81

Entry Ester Used 1 Iodoaryl

4h-o

Product 6

Yield (%)

5

1c

4j

6g (74 %)

6

1s

4k

6h (71 %)

7

1s

4l

6i (62 %)

8

1t

4k

6j (44%)

9

1u

4m

6k (64 %)

10

1aa

4h

6l (51 %)

Results and Discussion

82

Entry Ester Used 1 Iodoaryl

4h-o

Product 6

Yield (%)

11

1dd

4h

6m (71 %)

12

1dd

4n

-

13

1t

4n

-

14

1aa

4o

-

Presence of electron withdrawing groups facilitate cycloisomerization due to their inductive

effect154 as shown in Scheme 5.20.

Scheme 5.20. Ease of cycloisomerization due to the inductive effect of electron withdrawing

group.154

But no cycloisomerized product formation was observed when iodoaryl systems with

electron withdrawing groups, 4j and 4m, were used for coupling reaction (Table 5.18, entries

5 and 9). That could be rationalized on the basis of structural rigidity of arylated

Results and Discussion

83

propargylamides 6 that are too rigid to acquire the proper structural conformational changes

required for cycloisomerization.

During the development of synthetic strategy, the presence of Cu(I) catalysis in the reaction

mixture, was further utilized by extending the strategy to triazole ligation on the coupled

product 6 hence extending the methodology by the incorporation of click reaction as the

terminal step of the one-pot procedure (Scheme 5.20).

Scheme 5.20. Synthesis of triazole ligated coupled product 8 via chemoenzymatic one-pot

amidation-coupling-clicks sequence.

Outlook

84

6.0. Outlook

The successful integration of the concept of lipase catalyzed aminolysis with subsequent

metal catalyzed cycloaddition and coupling reactions in a consecutive one-pot manner,

validates the synergy of biocatalysis with chemical catalyzed processes for the development

of novel multicomponent methodologies. In this study, propargylamine (2) has been utilized

as a suitable motif for the generation of corresponding amide functionality with terminal

alkyne group for further modifications in one-pot fashion. Some of the other possibilities

utilizing alkyne moiety of propargylamide 3 for the syntheses of heterocyclic systems, have

been depicted in Scheme 6.1, below.

Scheme 6.1. Retrosynthetic analyses for the syntheses of a) fused ring triazoles, b)

Isoxazoles, c) Imidazoles, suggesting the integration of Novozyme® 435 catalysis for the

development of novel one-pot strategies.

One possibility might be the generation of fused ring triazole systems via click reaction

following the lipase catalyzed aminolysis (Scheme 6.1a). The optimized lipase catalyzed

aminolysis for propargylamide 3 generation can further be tuned to other catalyst systems

for the syntheses of heterocyclic systems. In this context, hypervalent iodine species (PIDA,

Outlook

85

PIFA) have been actively employed for the synthesis of isoxazole systems181 and this

methodology can be assimilated with lipase catalyzed aminolysis for the development of

metal free one-pot isoxazole syntheses (Scheme 6.1b). Synthesis of imidazoles have been

reported by hydroamination of propargylamides in the presence of zinc catalyst system

(Zn(OTf)2).182 Since the synergistic behaviour of lipases with zinc catalyst systems has not

yet been explored, therefore, this synthetic void also provides the opportunity to study the

integration of lipase catalysis with zinc catalyzed hydroamination of alkyne moiety of

propargylamides 3 leading to imidazole system (Scheme 6.1c).

Experimental

86

7.0. Experimental

7.1. General Considerations

All enzyme catalyzed and coupling reactions were performed in flame dried Schlenk tubes.

The enzyme catalyzed reactions were carried out under normal atmospheric conditions in an

incubating shaker IKA® KS 4000i, whereas the Sonogashira coupling reactions were

executed under argon atmosphere. MTBE was dried over 3 Å molecular sieves prior to use.

The reaction progress was monitored qualitatively by thin layer chromatography using silica

gel layered aluminium foil (60 F254 Merck, Darmstadt). The detection was made by

irradiating under UV light of wavelength 254 and 366 nm or stained with KMnO4, ninhydrin

and molybdate solution (saturated ethanolic solution of molybdatophosphoric acid). Crude

mixtures were absorbed on Celite® 545 (0.02-0.20 mm, Carl Roth GmbH Co.KG) for column

chromatography through flash technique. All the chemicals that have not been synthesized,

were purchased from Sigma Aldrich, Alfa Aesar and ACROS and were used as received

without any further purification. The 1H-NMR and 13C-NMR spectra were measured on the

device Avance DRX 500 , AV or AV III 600 III 300 Bruker. Chemical shifts are given in ppm

(δ) and were referenced to the internal solvent signal: d6-acetone (1H δ 2.05 13C δ 29.9),

CDCl3 (1H δ 7.26 , 13C δ 77.2 ), d6-DMSO (1H δ 2.50 13C δ 39.5 ). Multiplicities are stated as:

s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), dq

(doublet of quartet), m (multiplet), br m (broad multiplet). Coupling constants (J) are given in

Hz. The assignment of the primary (CH3), secondary (CH2), tertiary (CH) and quaternary

carbon nuclei (Cquat), was made using DEPT-135 spectra. The mass spectrometric

investigations were carried out in the Department of Mass Spectrometry of Inorganic and

Organic Chemistry of the University of Düsseldorf. The IR spectra were recorded with a

Bruker Vector 22 FT-IR or Shimadzu IRAffinity-1. The intensities of the IR bands were

abbreviated as w (weak), m (medium), s (strong). Melting points (uncorrected) were

measured using Reichert Thermovar (PeakTeck®). Combustion analyses were measured on

a Perkin Elmer Series II Analyser 2400 in the Institute for Pharmaceutical and Medicinal

Chemistry Heinrich-Heine University, Düsseldorf.

Experimental

87

7.2. Synthesis of Starting Materials

Ester starting materials have been synthesized according to the respective methods

reported in literature.

Methyl 2-(benzyloxy) acetate (1f),183 methyl 2-(phenylamino)acetate (1h),184 methyl 2-

(phenylthio)acetate (1i),185 methyl 2-(benzylthio)acetate (1j),185 methyl 3-(4-(2-methoxy-2-

oxoethoxy)phenyl)propanoate (1k),186 (R)-methyl 2-((t-butoxycarbonyl)amino)propanoate

(1m),187 (R)-methyl 2-(2,2,2-trifluoroacetamido)propanoate (1n),188 (R)-methyl 2-((benzyloxy)

carbonyl)amino)propanoate (1o),189 (S)-methyl 2-((benzyloxy)carbonyl)amino)

propanoate (1p),189 methyl 2-(2,2,2-trifluoroacetamido)acetate (1dd).188

7.2.1. Methyl 2-(benzyloxy)acetate (1f)183

A solution of 2-(benzyloxy) acetic acid (2.0 g, 12.00 mmol) in MeOH (40.0 mL), was cooled

to 4 °C in an ice bath. Thionyl chloride (1.7 g, 14.00 mmol) was added drop wise maintaining

the temperature below 8 °C. The resulting solution was stirred for 30 minutes at 4 °C and for

2.5 h at room temperature. The solvent was evaporated under reduced pressure and the

resulting residue was dissolved in ethylacetate (20.0 mL) and washed with saturated

aqueous NaHCO3 solution (20.0 mL). The aqueous layer was extracted with additional

portion of ethylacetate (2 x 10.0 mL). The combined organic layers were washed with brine

(20.0 mL), dried with anhydrous Na2SO4, filtered and evaporated under reduced pressure to

get 1.62 g (75 %) of title compound as colorless clear liquid.

1H-NMR (300 MHz, CDCl3): δ = 3.76 (s, 3 H), 4.11 (s, 2 H), 4.64 (s, 2 H), 7.27-7.38 (m, 5 H).

13C-NMR (75 MHz, CDCl3): δ = 51.2 (CH3), 61.1 (CH2), 73.37 (CH2), 128.0 (CH), 128.1 (CH),

128.5 (CH), 137.0 (Cquat), 170.8 (Cquat). EI-MS: m/z (%) = 180 (M+, 1.3), 121 (C8H9O+, 4.9),

105 (C4H9O3+, 3.6), 107 (C7H7O

+, 79.9), 91 (C7H7+, 100), 89 (C3H5O3

+, 5.4). Anal. calcd. for

C10H12O3 (180.2): C 66.65, H 6.71. Found C 66.51, H 6.80.

Experimental

88

7.2.2. Methyl 2-(phenylamino)acetate (1h)184

In a 50 mL round bottom flask containing acetone (10.0 mL), were added aniline (1.03 g,

11.10 mmol), and potassium carbonate (2.3 g, 16.70 mmol). The reaction mixture was

allowed to stir at 60 °C for 1 h after which methyl bromoacetate (1.85 g, 12.10 mmol), was

added drop wise to the suspension and the mixture was stirred for 20 h at 60 °C. After

20 h, the reaction mixture was filtered and diluted with ethylacetate (20.0 mL) for

subsequent washing with water (10.0 mL) and brine (10.0 mL). The organic layer was dried

with anhydrous Na2SO4 followed by silica gel column chromatography using n-

hexane:ethylacetate (4:1) as eluent to get 1.23 g (67 %) of title compound as greyish brown

solid.

1H-NMR (300 MHz, CDCl3): δ = 3.79 (s, 3 H), 3.93 (s, 2 H), 4.29 (br s, 1 H), 6.62 (d, 3J = 8.5

Hz, 2 H), 6.76 (m, 1 H), 7.20 (m, 2 H). 13C-NMR (75 MHz, CDCl3): δ = 45.8 (CH2), 52.4

(CH3), 113.1 (CH), 118.4 (CH), 129.5 (CH), 147.1 (Cquat), 171.8 (Cquat). EI-MS: m/z (%) = 165

(M+, 16.7), 106 (C8H10N+, 100), 77 (C6H5

+, 30.3). Anal. calcd. for C9H11O2N (165.2): C 65.44

N 8.48, H 6.71. Found C 65.63 N 8.30 H 6.52.

7.2.3. Methyl 2-(phenylthio)acetate (1i)185

In a 50 mL round bottom flask, a mixture of thiophenol (1.11 g, 10.00 mmol), triethylamine

(1.01 g, 10.0 mmol), methyl bromoacetate (1.53 g, 10.00 mmol), and benzene (13.0 mL) was

stirred at room temperature for 4 h after which the reaction mixture was filtered and diluted

with ethylacetate (20.0 mL) followed by subsequent washing with water (10.0 mL) and brine

(10.0 mL). The organic layer was dried with anhydrous Na2SO4 followed by silica gel column

Experimental

89

chromatography (n-hexane:ethylacetate 10:1) to get 1.39 g (96 %) of title compound as

colorless liquid.

1H-NMR (300 MHz, CDCl3): δ = 3.65 (s, 2 H), 3.71 (s, 2 H), 7.23 (t, 3J = 7.5 Hz, 1 H), 7.30 (t,

3J = 7.5, 2 H), 7.41 (d, 3J = 7.3 Hz, 2 H).13C-NMR (75 MHz, CDCl3): δ = 52.6 (CH3), 36.6

(CH2), 127.1 (CH), 129.2 (CH), 130.0 (CH), 135.0 (Cquat), 170.2 (Cquat). EI-MS: m/z (%) = 182

(M+, 32.9), 123 (C7H7S+, 100), 109 (C6H5S

+, 17.8), 77 (C6H5+, 30.5). Anal. calcd. for

C9H10O2S (182.2): C 59.32 H 5.53. Found C 59.26 H 5.35.

7.2.4. Methyl 2-(benzylthio)acetate (1j)185

Benzyl mercaptan (0.62 g, 5.00 mmol), triethylamine (0.51 g, 5.00 mmol) and methyl bromo-

acetate (0.76 g, 5.00 mmol) in benzene (7.5 mL), were stirred at room temperature for 4 h in

a 50 mL round bottom flask. After the aqueous work up, same as mentioned for 1h and 1i,

and drying with anhydrous Na2SO4, the product was purified by silica gel chromatography (n-

hexane:ethylacetate (10:1)) to get 0.77 g (78 %) of title compound as colorless liquid.

1H-NMR (300 MHz, CDCl3): δ 3.06 (s, 2 H), 3.69 (s, 3 H), 3.90 (s, 2 H), 7.2 -7.31 (m, 5 H).

13C-NMR (75 MHz, CDCl3): δ 32.2 (CH2), 36.5 (CH2), 52.5 (CH3), 127.4 (CH), 128.7 (CH),

129.3 (CH), 137.3 (Cquat), 171.0 (Cquat).

7.2.5. Methyl 3-(4-(2-methoxy-2-oxoethoxy)phenyl)propanoate (1k)186

To a mixture of methyl 3-(4-hydroxyphenyl)propionate (2.70 g, 15.00 mmol) and K2CO3 (8.09

g, 57.00 mmol), was added methyl bromoacetate (4.58 g, 30.00 mmol) and the resulting

mixture was allowed to stir at 70 °C for 30 h. After 30 h of reaction time, the reaction mixture

was filtered and acetone was removed under vacuum. The residue was redissolved in water

Experimental

90

and extracted with chloroform (3 x 20.0 mL). The combined organic layers were dried with

anhydrous Na2SO4 and solvent was removed under vacuum to get crude solid that was

subjected to vacuum distillation with the gradual increase of temperature to get the 1.44 g

(38 %) of pure product (bp. 210-213 °C) as low melting colorless solid.

1H-NMR (300 MHz, CDCl3): δ = 2.59 (t, 3J = 7.4 Hz, 2 H), 2.89 (t, 3J = 7.7 Hz, 2 H), 3.66 (s, 1

H), 3.80 (s, 3 H), 4.60 (s, 2 H), 6.83 (d, 3J = 8.6 Hz, 2 H) , 7.11 (d, 3J = 8.4 Hz, 2 H). 13C-

NMR (75 MHz, CDCl3): δ = 30.6 (CH2), 36.4 (CH2), 52.2 (CH3), 52.8 (CH3), 65.8 (CH2), 115.3

(CH), 129.9 (CH), 134.4 (Cquat), 156.9 (Cquat), 170.0 (Cquat), 173.9 (Cquat). GC-MS: m/z (%) =

252 (M+, 21.1), 179 (C10H11O3+, 100), 193 (C11H13O3

+, 6.3), 221 ([M - CH3O]+, 3.3), 91 (C7H7+,

37.6), 77 (C6H5+, 30.8), 59 (C2H3O2

+, 21.4). Anal. calcd for C13H16O5 (252.3): C 61.90 H 6.39.

Found C 61.96 H 6.38.

7.2.6. (R)-Methyl 2-((t-butoxycarbonyl)amino)propanoate (1m)187

To a suspension of D-alanine (0.89 g, 10.00 mmol) in ice cooled methanol (17.0 mL) in a

50 mL round bottom flask, thionyl chloride (1.69 g, 14.20 mmol) was drop wise added

maintaining the temperature between 0-8 °C. After the complete drop wise addition of thionyl

chloride, the reaction mixture was allowed to stir for 20 min at 0 °C and then at room

temperature overnight. After overnight stirring, the reaction mixture was heated to 80 °C for

3 h and solvent was removed under reduced pressure to get viscous light yellow colored

residue that was then redissolved in saturated aqueous NaHCO3 solution (24.0 mL) followed

by drop wise addition of 1,4-dioxane (25.0 mL) solution of Boc-anhydride (3.00 g, 13.70

mmol). The reaction mixture was allowed to run for 12 h at room temperature after which it

was filtered and extracted with ethylacetate (3 x 15.0 mL). The combined organic layers

were dried with anhydrous Na2SO4. The evaporation of organic solvent under reduced

pressure followed by drying under vacuum, gave 1.48 g (72 %) of title compound as

colorless viscous liquid.

Experimental

91

1H-NMR (300 MHz, CDCl3): δ = 1.39 (d, 3J = 7.2 Hz, 3 H), 1.45 (s, 9 H), 3.75 (s, 3 H), 4.33

(m, 1 H), 5.05 (br s, 1 H). 13C-NMR (75 MHz, CDCl3): δ = 18.4 (CH3), 28.0 (CH3), 48.9 (CH),

52.0 (CH3), 79.6 (Cquat), 154.8 (Cquat), 173.6 (Cquat). GC-MS: m/z (%) = 203 (M+, 0.2), 188

(C8H14NO4+, 188.0), 144 (C7H14NO2

+, 18.1), 130 (C5H8NO3+, 3.7), 116 (C5H10NO2

+, 2.3), 102

(C4H8NO2+, 12.7), 88 (C4H8O2

+, 23.1), 70 (5.3), 57 (C4H9+, 100).

7.2.7. (R)-Methyl 2-(2,2,2-trifluoroacetamido)propanoate (1n)188

To a suspension of D-alanine (0.89 g, 10.00 mmol) in ice cooled methanol (17.0 mL) in a 50

mL round bottom flask, thionyl chloride (1.69 g, 14.20 mmol) was drop wise added

maintaining the temperature between 0-8 °C. After the complete drop wise addition of thionyl

chloride, the reaction mixture was allowed to stir for 20 min at 0 °C and then at room

temperature overnight. Colorless liquid was obtained after evaporating the solvent under

reduced pressure. To this colorless liquid, was added DCM (20.0 mL) in an ice bath followed

by slow addition of triethylamine (3.03 g, 30.00 mmol) maintaining the temperature below

5 °C. After the complete addition of triethylamine, trifluoroacetic anhydride (2.31 g, 11.00

mmol) was added drop wise in the reaction mixture maintaining the temperature below 5 °C.

The reaction mixture was allowed to run for 30 min on ice bath and for 5 h at room

temperature after which water (15.0 mL) was added into it for subsequent extraction with

ethylacetate (3 x 15.0 mL) and washing with 2N HCl solution. The combined organic layers

were dried with anhydrous Na2SO4. Purification by silica gel column chromatography (n-

hexane:ethylacetate (2:1)) furnished 1.25 g (63 %) of the title compound as viscous brown

liquid.

1H-NMR (300 MHz, CDCl3): δ = 1.51 (d, 3J = 7.2 Hz, 3 H), 3.81 (s, 3 H), 4.61 (m, 1 H), 6.99

(br s, 1 H). 13C-NMR (75 MHz, CDCl3): δ = 17.6 (CH3), 48.3 (CH3), 52.8 (CH), 115.3 (d, 1J =

287.6 Hz, Cquat), 156.6 (d, 2J = 37.9 Hz, Cquat), 171.7 (Cquat). GC-MS: m/z (%) = 199 (M+, 0.9),

184 (C5H5F3NO3+, 1.1), 167 (0.9), 140 (100), 129 (2.4), 102 (C4H8NO2

+, 8.4), 113 (3.4), 92

(10.7), 70 (23.3), 69 (CF3+, 48.7), 59 (C2H3O2

+, 13.9).

Experimental

92

7.2.8. (R)-Methyl 2-((benzyloxy)carbonyl)amino)propanoate (1o)189

To a suspension of D-alanine (0.89 g, 10.00 mmol) in ice cooled methanol (17.0 mL) in a 50

mL round bottom flask, thionyl chloride (1.69 g, 14.20 mmol) was drop wise added

maintaining the temperature between 0-8 °C. After the complete drop wise addition of thionyl

chloride, the reaction mixture was allowed to stir for 20 minutes at 0 °C and then at room

temperature overnight after which it was heated to 80 °C for 3 h. Removal of the solvent

under reduced pressure gave the respective methyl ester as colorless liquid that was then

dissolved in saturated aqueous NaHCO3 solution (20.0 mL) followed by the addition of

benzyl chloroformate (1.70 g, 10.00 mmol) under vigorous stirring overnight. The reaction

mixture was extracted with diethylether (3 x 15.0 mL) and washed with 2N HCl (20.0 mL)

solution. The combined organic layers were dried with anhydrous Na2SO4. Purification by

silica gel column chromatography (n-hexane:ethylacetate (3:1)) furnished 1.76 g (74 %) of

the title compound as colorless waxy solid.

1H-NMR (300 MHz, CDCl3): δ = 1.41 (d, 3J = 7.2 Hz, 3 H), 3.74 (s, 3 H), 4.39 (m, 1 H), 5.11

(s, 2 H), 5.34 (br s, 1 H), 7.31-7.36 (m, 5 H). 13C-NMR (75 MHz, CDCl3): δ = 18.4 (CH3), 43.3

(CH3), 52.2 (CH), 66.7 (CH2), 127.8 (CH), 127.9 (CH), 128.2 (CH), 136.0 (Cquat), 155.3

(Cquat), 173.2 (Cquat). GC-MS: m/z (%) = 237 (M+, 2.6), 178 (C10H12NO2+, 12.6), 179 (1.3), 135

(C8H6O2+, 1.6), 134 (15.3), 108 (C7H8O

+, 36.5), 109 (C7H9O+, 2.7), 91 (C7H7

+, 100), 92 (7.5),

102 (C4H8NO2+, 1.7), 59 (C2H3O2

+, 1.4), 89 (C4H9O2+, 3.7), 70 (1.8), 79 (3.3), 65 (C5H5

+, 5.8),

59 (1.4), 51 (1.4), 44 (2.4).

Experimental

93

7.2.9. (S)-Methyl 2-((benzyloxy)carbonyl)amino)propanoate (1p)189

L-alanine methyl ester (1.40 g, 10.00 mmol) was dissolved in aqueous saturated NaHCO3

solution (20.0 mL) in a 50 mL round bottom flask. Ethylacetate (20.0 mL) was added to the

reaction mixture followed by the addition of benzyl chloroformate (1.70 g, 10.00 mmol) and

reaction was allowed to run overnight. After the overnight stirring, the organic layer was

separated and aqueous layer was extracted with ethylacetate (3 x 10.0 mL). The combined

organic layers were washed with 2N HCl solution (15.0 mL) and dried with anhydrous

Na2SO4. Evaporation of the solvent yielded 1.96 g (83 %) of the title compound as colorless

solid.

1H-NMR (300 MHz, CDCl3): δ = 3.75 (s, 3 H), 4.39 (br m, 1 H), 5.11 (s, 2 H), 5.31 (br s, 1 H),

7.29-7.42 (br m, 5 H).13C-NMR (75 MHz, CDCl3): δ = 18.8 (CH3), 49.7 (CH3), 52.6 (CH), 67.1

(CH2), 128.2 (CH), 128.3 (CH), 128.7 (CH), 136.4 (Cquat), 155.7 (Cquat). GC-MS: m/z (%) =

237 (M+, 1.1), 178 (C10H12NO2+, 2.3), 148 (C5H10NO4

+, 1.7), 134 (5.7), 108 (C7H8O+, 21.5),

109 (C7H9O+, 1.7), 91 (C7H7

+, 100), 77 (C6H5+, 7.3), 65 (C5H5

+, 5.9).

7.2.10. Methyl 2-(2,2,2-trifluoroacetamido)acetate (1dd)188

To a mixture of glycine methyl ester hydrochloride (1.26 g, 10.00 mmol) in DCM (20.0 mL),

was added triethylamine (3.04 g, 30.00 mmol) at 0 °C followed by addition of trifluoroacetic

anhydride (2.31 g, 11.00 mmol) maintaining the temperature between 0-8 °C. After the

complete addition of trifluoroacetic anhydride, the reaction mixture was allowed to stir at

room temperature for 5 h after which the reaction mixture was treated with water (20.0 mL)

and extracted with ethylacetate (3 x 15.0 mL). The combined organic layers were washed

with 2N HCl solution (20.0 mL) and dried with anhydrous Na2SO4. The solvent was removed

Experimental

94

under reduced pressure and 1.12 g (61 %) of the title compound was obtained as colorless

liquid after purification by silica gel column chromatography (n-hexane:ethylacetate (3:1)).

1H-NMR (300 MHz, CDCl3): δ = 3.76 (s, 3 H), 4.07 (d, 3J = 5.1, 2 H), 6.83 (br s, 1 H). 13C-

NMR (75 MHz, CDCl3): δ = 39.6 (CH2), 48.7 (CH3), 117.6 (d, 1J = 286.8 Hz, Cquat), 156.9 (d,

2J = 37.5 Hz, Cquat), 169.8 (Cquat). GC-MS: m/z (%) = 185 (M+, 0.6), 154 ([M - CH3O]+, 3.2),

126 (C3H3F3NO+, 100), 106 (4.89), 88 ([M - C2F3O]+, 13.9), 69 (CF3+, 48.6), 59 (C2H3O2

+,

15.7).

7.3. General Procedure for CAL-B (Novozyme® 435) Catalyzed Aminolysis

To a solution of 55 mg (1.00 mmol) of propargylamine (2), in dry MTBE (2.0 mL) in a

screwcap Schlenk vessel, were added 1.20 mmol of the methyl ester 1 (experimental details

mentioned in Table 7.1) and Novozyme® 435 (50 % w/w of corresponding ester substrate 1

used) and the reaction was shaken in an incubating shaker at 45 °C for 4 or 24 h (depending

upon the nature of ester 1 used). After the given reaction time (Table 7.1), the enzyme

beads were filtered off and then the filtered reaction mixture was subjected to silica gel

column chromatography using n-hexane:ethylacetate as eluent to obtain the pure product 3.

Table 7.1. Experimental details for Candida antarctica lipase B (Novozyme® 435) catalyzed

aminolysis by propargylamine (2, 55.0 mg,1.00 mmol) at 45 °C.

Entry Ester Substrate 1 t [h] Propargylamide 3

1 233 mg (1.20 mmol) of 1a 24 148 mg (68 % ) of 3a

2 197 mg (1.20 mmol) of 1b 24 116 mg (62 %) of 3b

3 180 mg (1.20 mmol) of 1c 24 126 mg (73 % ) of 3c

4 199 mg (1.20 mmol) of 1e 4 165 mg (87%) of 3e

5 216 mg (1.20 mmol) of 1f 4 175 mg (86 %) of 3f

6 235 mg (1.20 mmol) of 1g 4 208 mg (95 %) of 3g

7 198 mg (1.20 mmol) of 1h 4 152 mg (81 %) of 3h

8 219 mg (1.20 mmol) of 1i 4 158 mg (77 %) of 3i

9 236 mg (1.20 mmol) of 1j 4 180 mg (82 %) of 3j

10 303 mg (1.20 mmol) of 1k 4 220 mg (80%) of 3k

11 239 mg (1.20 mmol) of 1n 4 138 mg (62 %) of 3n

12 285 mg (1.20 mmol) of 1o 4 213 mg (82 %) of 3o

13 285 mg (1.20 mmol) of 1p 4 208 mg (80 %) of 3p

14 316 mg (1.20 mmol) of 1q 24 195 mg (68 %) of 3q

15 304 mg (1.20 mmol) of 1r 24 146 mg (53 %) of 3r

Experimental

95

Entry Ester Substrate 1 t [h] Propargylamide 3

16 151 mg (1.20 mmol) of 1s 24 106 mg (71 %) of 3s

17 171 mg (1.20 mmol) of 1t 24 127 mg (77 %) of 3t

18 205 mg (1.20 mmol) of 1u 4 126 mg (70 %) of 3u

19 192 mg (1.20 mmol) of 1aa 24 147 mg (80 %) of 3aa

7.4. Analytical Data of Propargylamides 3

7.4.1. 3-(4-Methoxyphenyl)-N-prop-2-yn-1-yl)propanamide (3a)

148 mg (0.68 mmol, 68 %) of 3a as colorless crystalline substance was obtained after

purification by silica gel column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 88 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.21 (t, 4J = 2.6 Hz, 1 H), 2.46 (t, 3J = 7.7 Hz,

2 H), 2.90 (t, 3J = 7.7 Hz, 2 H), 3.77 (s, 3 H), 4.01 (dd, 3J = 5.2 Hz, 4J = 2.5 Hz, 2 H), 5.70 (br

s, 1 H), 6.82 (d, 3J = 8.6 Hz, 2 H), 7.10 (d, 3J = 8.6 Hz, 2 H). 13C-NMR (75 MHz, CDCl3): δ =

29.3 (CH2), 30.8 (CH2), 38.6 (CH2), 55.4 (CH3), 71.7 (CH), 79.6 (Cquat), 114.1 (CH), 129.4

(CH), 132.8 (Cquat), 158.2 (Cquat), 172.0 (Cquat). EI-MS: m/z (%) = 217 (M+, 5.8), 121 ([M -

C5H6NO]+, 100), 134 (12.3), 180 (C10H14NO2+, 21.8), 77 (C6H5

+, 7.6), 91 (C7H7+, 9.3), 65

(C5H5+, 3.6). IR (ATR) [cm-1] = 3068 (w), 2962 (w), 2922 (w), 2860 (w), 2835 (w), 2673 (w),

1634 (s), 1612 (w), 1544 (m), 1512 (s), 1456 (w), 1421 (w), 1375 (w), 1340 (w), 1301 (w),

1296 (w), 1240 (s), 1228 (s), 1178 (m), 1111 (w), 1087 (w), 1028 (s), 923 (w), 887 (w), 821

(w), 802 (m), 771 (w), 721 (s), 688 (s), 615 (w). Anal. calcd. for C13H15NO2 (217.3): C 71.87,

H 6.96, N 6.45. Found: C 71.87, H 6.94, N 6.24.

Experimental

96

7.4.2. 3-Phenyl-N-(prop-2-yn-1-yl)propanamide (3b)

116 mg (0.62 mmol, 62 %) of 3b as colorless crystalline substance was obtained after

purification by silica gel column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 69 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.20 (t, 4J = 2.5 Hz, 1 H), 2.50 (t, 3J = 7.6 Hz, 2

H), 2.97 (t, 3J = 7.6 Hz, 2 H), 4.01 (dd, 3J = 5.3 Hz, 4J = 2.6 Hz, 2 H), 5.74 (br s, 1 H), 7.18-

7.21 (m, 3 H), 7.27-7.29 (m, 2 H). 13C-NMR (75 MHz, CDCl3): δ = 29.3 (CH2), 31.6 (CH2),

38.2 (CH2), 71.7 (CH), 79.6 (Cquat), 126.4 (CH), 128.4 (CH), 128.7 (CH), 140.8 (Cquat), 171.9

(Cquat). EI-MS: m/z (%) = 188 ([M + H]+, 25.1), 187 (M+, 97.4), 186 ([M - H]+, 18.6), 170 (8.3),

169 (32.0), 158 (5.2), 146 (8.1), 144 (13.8), 143 (18.9), 141 (16.6), 131 (C9H7O+, 12.9),

129 (C9H5O+, 33.0), 128 (C9H4O

+, 30.0), 117 (4.4), 116 (3.3), 115 (5.5), 110 ([M - C6H5]+,

9.3), 105 (C8H9+, 59.3), 104 (C8H8

+, 38.3), 96 ([M - C7H7]+, 76.0), 91 (C7H7

+, 100), 82 (4.6),

65 (C5H5+, 15.5), 55 (C3H5N

+, 16.0), 51 (C3HN+, 12.4), 39 (C3H3+, 25.8). IR (ATR) [cm-1] =

3055 (w), 2988 (w), 2951 (w), 2901 (w), 2886 (w), 2835 (w), 2581 (w), 1732(s), 1599 (w),

1585 (w), 1558 (w), 1437 (m), 1368 (m), 1317 (w), 1261 (w), 1223 (m), 1217 (m), 1179 (m),

1138 (m), 1076 (w), 1057 (w), 1028 (w), 984 (w), 957 (w), 916 (w), 870 (w), 839 (w), 820 (w),

754 (m), 741 (s), 694 (s), 602 (w). Anal. calcd. for C12H13NO (187.2): C 76.98, H 7.00,

N 7.48. Found: C 76.89, H 7.01, N 7.49.

Experimental

97

7.4.3. 2-Phenyl-N-(prop-2-yn-1-yl)acetamide (3c)

126 mg (0.73 mmol, 73 %) of 3c as colorless crystalline substance was obtained after

purification by silica gel column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 71 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.18 (t, 4J = 2.3 Hz, 1 H), 3.59 (s, 2 H), 4.01 (dd,

3J = 5.3, 4J = 2.6, 2 H), 5.67 (br s, 1 H), 7.25 (m, 1 H), 7.27-7.39 (m, 4 H). 13C-NMR (75 MHz,

CDCl3): δ = 29.4 (CH2), 43.6 (CH2), 71.7 (CH), 79.5 (Cquat), 127.6 (CH), 129.2 (CH), 129.6

(CH), 134.5 (Cquat), 170.7 (Cquat). EI-MS: m/z (%) = 174 ([M + H]+, 3.6), 173 (M+, 16.7), 172

([M - H]+, 1.1), 119 [C8H7O+, 1.8), 96 (C5H6NO+, 3.2), 91 (C7H7

+, 100), 82 (C4H4NO+, 10.4),

77 (C6H5+, 1.3), 65 (C5H5

+, 16.3), 43 (C2H3O+, 3.1), 39 (C3H3

+, 22.1). IR (ATR) [cm-1] =

3034 (w), 2972 (w), 2922 (w), 2866 (w), 2804 (w), 1664 (w), 1627 (m), 1539 (m), 1490 (m),

1448 (m), 1429 (w), 1394 (w), 1363 (w), 1330 (w), 1315 (w), 1292 (w), 1263 (w), 1199 (w),

1155 (w), 1103 (w), 1082 (w), 1070 (w), 1028 (w), 1002 (w), 920 (w), 906 (w), 800 (w), 769

(w), 731 (w), 690 (s), 677 (s), 650 (m), 607 (m). Anal. calcd. for C11H11NO (173.2): C 76.28,

H 6.40, N 8.09. Found C 76.20, H 6.25, N 7.81.

7.4.4. 2-Phenoxy-N-(prop-2-yn-1-yl) acetamide (3e)

165 mg (0.87 mmol, 87%) of 3e as colorless crystalline substance was obtained after

purification by silica gel column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 102 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.26 (t, 4J = 2.6 Hz, 1 H), 4.15 (dd, 3J = 5.5 Hz,

4J = 2.6 Hz, 2 H), 4.51 (s, 2 H), 6.84 (br s, 1 H), 6.92 (dd, 3J = 8.6 Hz, 4J = 0.8 Hz, 2 H), 7.03

(tt, 3J = 7.4 Hz, 4J = 0.8 Hz, 1 H), 7.32 (dd, 3J = 8.7 Hz, 7.4 Hz, 2 H). 13C-NMR (75 MHz,

CDCl3): δ = 28.8 (CH2), 67.4 (CH2), 72.0 (CH), 79.1 (Cquat), 114.8 (CH), 122.4 (CH), 129.9

(CH), 157.2 (Cquat), 168.1 (Cquat). EI-MS: m/z (%) = 190 ([M + H]+, 13.8), 189 (M+, 100), 108

Experimental

98

([M - C4H3NO]+, 53.9), 107 (C7H7O+, 24.4), 96 ([M - C6H5O]+, 26.1), 77 (C6H5

+, 54.6),

55 (C3H5N+, 8.8). IR (ATR) [cm-1] = 3039 (w), 2914 (w), 1658 (m), 1598 (w), 1527 (m),

1494 (m), 1442 (m), 1413 (w), 1363 (w), 1350 (w), 1284 (w), 1170 (s), 1080 (w), 1062 (m),

1028 (w), 840 (w), 750 (s), 675 (m), 648 (m), 609 (m). Anal. calcd. for C11H11NO2 (189.2):

C 69.83, H 5.89, N 7.40. Found: C 69.86, H 5.69, N 7.36.

7.4.5. 2-(Benzyloxy)-N-(prop-2-yn-1-yl)acetamide (3f)

175 mg (0.86 mmol, 86 %) of 3f was obtained as colorless liquid after purification by silica

gel column chromatography (n-hexane:ethylacetate (2:1)).

1H-NMR (300 MHz, CDCl3): δ = 2.24 (t, 4J = 2.6 Hz, 1 H), 4.01 (s, 2 H), 4.08 (dd, 3J = 5.6 Hz,

4J = 2.6 Hz, 2 H), 4.58 (s, 2 H), 6.77 (br s, 1 H), 7.31-7.42 (m, 5 H). 13C-NMR (75 MHz,

CDCl3): δ = 28.6 (CH2), 69.5 (CH2), 71.8 (CH), 73.8 (CH2), 79.4 (Cquat), 128.2 (CH), 128.5

(CH), 128.8 (CH), 136.8 (Cquat), 169.4 (Cquat). EI-MS: m/z (%) = 91 (C7H7+, 100), 96 ([M -

C7H7O]+, 62.3), 77 (C6H5+, 8.2), 55 (C3H5N

+, 18.0). IR (ATR) [cm-1] = 3032 (w), 2912 (w),

2862 (w), 2121 (w), 1880 (w), 1813 (w), 1662 (s), 1519 (s), 1454 (w), 1442 (w), 1421 (w),

1340 (w), 1278 (w), 1259 (w), 1207 (w), 1099 (s), 1026 (w), 1002 (w), 966 (w), 925 (w), 912

(w), 850 (w), 819 (w), 738 (m), 698 (s), 667 (m), 650 (m). Anal. calcd. for C12H13NO2 (203.2):

C 70.92, H 6.47, N 6.89. Found: C 70.97, H 6.47, N 6.95.

7.4.6. 2-(4-Hydroxyphenoxy)-N-(prop-2-yn-1-yl)propanamide (3g)

208 mg (0.95 mmol, 95 %) of 3g was obtained as white crystalline solid after purification by

silica gel column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 112 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 1.32 (d, 3J = 6.6 Hz, 3 H), 3.06 (t, 4J = 2.5

Hz, 1 H), 3.85 (m, 2 H), 4.50 (q, 3J = 6.6 Hz, 1 H), 6.61-6.65 (m, 2 H), 6.71-6.74 (m, 2 H),

8.46 (t, 3J = 5.6 Hz, 1 H), 8.98 (s, 1 H). 13C-NMR (75 MHz, d6-DMSO): δ = 18.6 (CH3), 27.8

Experimental

99

(CH2), 72.7 (CH), 74.6 (CH), 81.1 (Cquat), 115.7 (CH), 116.8 (CH), 149.9 (Cquat), 151.8 (Cquat),

171.4 (Cquat). EI-MS: m/z (%) = 220 ([M + H]+, 9.2), 219 (M+, 65.6), 137 ([M - C4H4NO]+,

88.6), 110 ([M - C6H5O2]+, 100), 109 (C6H5O2

+, 11.5), 111 (C6H7O2+, 8.6), 93 (C6H5O

+, 8.1),

82 (C4H4NO+, 19.2), 81 (C4H3NO+, 22.8), 65 (C5H5+, 14.8), 56 (C3H6N

+, 5.1), 55 (C3H5N+,

15.9), 39 (C3H3+, 21.3). IR (ATR) [cm-1] = 3296 (w) 3259 (m), 2976 (w), 2966 (w), 2931 (w),

2900 (w), 2819 (w), 2744 (w), 2692 (w), 2609 (w), 2567 (w), 2505 (w), 2476 (w), 2434 (w),

2125 (w), 1988 (w), 1651 (s), 1604 (w), 1556 (m), 1508 (s), 1463 (w), 1440 (w), 1425 (w),

1373 (w), 1344 (w), 1303 (w), 1220 (s), 1207 (s), 1176 (w), 1141 (m), 1118 (w), 1093 (m),

1058 (m), 1047 (w), 1008 (w), 941 (w), 916 (w), 902 (w), 850 (w), 823 (m), 802 (w), 783 (w),

759. (s), 711 (w), 688 (m), 640 (w). Anal. calcd. for C12H13NO3 (219.2): C 65.74, H 5.98,

N 6.39. Found C 65.58, H 6.06, N 6.48.

7.4.7. 2-(Phenylamino)-N-(prop-2-yn-1-yl)acetamide (3h)

152 mg (0.81 mmol, 81 %) of 3h was obtained as brownish yellow viscous liquid after

purification by silica gel column chromatography (n-hexane:ethylacetate (2:1)).

1H-NMR (300 MHz, CDCl3): δ = 2.25 (t, 4J = 2.6 Hz, 1 H), 3.88 (d, 3J = 5.4 Hz, 2 H), 4.13 (dd,

3J = 5.6 Hz, 4J = 2.6 Hz, 2 H), 4.37 (br s, 1 H), 6.69 (d, 3J = 7.6 Hz, 2 H), 6.90 (t, 3J = 7.4 Hz,

1 H), 7.01 (br s, 1 H), 7.27-7.32 (m, 2 H). 13C-NMR (75 MHz, CDCl3): δ = 29.0 (CH2), 49.0

(CH2), 71.7 (CH), 79.4 (Cquat), 113.4 (CH), 119.4 (CH), 129.6 (CH), 147.2 (Cquat), 170.6

(Cquat). EI-MS: m/z (%) = 189 ([M + H]+, 2.5), 188 (M+ , 19.9), 106 (C7H8N+, 100), 104 (3.0),

93 (1.1), 77 (C6H5+, 12.1), 51 (4.2), 39 (C3H3

+, 2.6). IR (ATR) [cm-1] = 3356 (w), 3263 (m),

3024 (w), 1643 (s), 1602 (m), 1514 (s), 1496 (m), 1458 (w), 1435 (w), 1423.47 (w), 1354 (w),

1338 (w), 1319 (m), 1261 (w), 1240 (w), 1226 (w), 1182 (w), 1155 (w), 1109 (w), 1078 (w),

1022 (w), 941 (w), 881 (w), 756 (s), 698 (m), 677 (m), 659 (m), 630. (m), 617 (w).

Anal. calcd. for C11H12N2O (188.2): C 70.19, H 6.43, N 14.88. Found: C 70.01, H 6.70,

N 14.75.

Experimental

100

7.4.8. 2-(Phenylthio)-N-(prop-2-yn-1-yl)acetamide (3i)

158 mg (0.77 mmol, 77 %) of 3i was obtained as colorless crystalline solid after purification

by silica gel column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 63 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.20 (t, 4J = 2.6 Hz, 1 H), 3.64 (s, 2 H), 4.04 (dd,

3J = 5.4 Hz, 4J = 2.5 Hz, 2 H), 6.99 (br s, 1 H), 7.19-7.25 (m, 1 H), 7.28-7.31(m, 4 H).

13C-NMR (75 MHz, CDCl3): δ = 29.6 (CH2), 37.6 (CH2), 71.9 (CH), 79.0 (Cquat), 127.0 (CH),

128.5 (CH), 129.5 (CH), 134.5 (Cquat), 167.8 (Cquat). EI-MS: m/z (%) = 206 ([M + H]+, 7.6),

205 (M+, 48.4), 204 (2.2), 172 (6.5), 162 (3.3), 148 (2.7), 123 (C7H7S+, 48.3), 124 (C7H8S

+,

48.7), 125 (6.2), 109 (C6H5S+, 15.3), 110 (7.0), 111 (1.3), 96 ([M - C6H5S]+, 100), 83 (1.1), 91

(17.0), 79 (5.8), 78 (12.8), 77 (C6H5+, 12.7), 65 (C5H5

+, 7.8), 45 (27.5), 39 (C3H3+, 18.2).

IR (ATR) [cm-1] = 3062 (w), 2958 (w), 2358 (w), 1649 (s), 1624 (w), 1583 (w), 1544 (m),

1508 (w), 1479 (m), 1452 (w), 1436 (w), 1404 (w), 1354 (w), 1332 (w), 1309 (w), 1234 (w),

1215 (m), 1186 (w), 1165 (w), 1114 (w), 1089 (w), 1072 (w), 1022 (m), 937 (w), 887 (w), 783

(w), 736 (s), 646 (m), 615 (w). Anal. calcd. for C11H11NOS (205.3): C 64.36, H 5.40, N 6.82.

Found: C 64.11, H 5.54, N 6.83.

7.4.9. 2-(Benzylthio)-N-(prop-2-yn-1-yl)acetamide (3j)

180 mg (0.82 mmol, 82 %) of 3j was obtained as colorless crystalline solid after purification

by silica gel column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 57 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.25 (t, 4J = 2.6 Hz, 1 H), 3.14 (s, 2 H), 3.73 (s, 2

H), 3.96 (dd, 3J = 5.4 Hz, 4J = 2.6 Hz, 2 H), 6.83 (br s, 1 H), 7.27-7.36 (m, 5 H). 13C-NMR (75

MHz, CDCl3): δ = 29.5 (CH2), 35.2 (CH2), 37.2 (CH2), 71.9 (CH), 79.3 (Cquat), 127.6 (CH),

128.9 (CH), 129.1 (CH), 137.0 (Cquat), 168.3 (Cquat). EI-MS: m/z (%) = 220 ([M + H]+, 2.6),

219 (M+, 11.1), 123 (C7H7S+, 12.9), 121 (C7H5S

+, 2.9), 97 (C5H7NO+, 100), 96 (C5H6NO+,

52.2), 98 (C5H8NO+, 7.5), 91 (C7H7+, 69.7), 89 (2.8), 77 (C6H5

+, 2.6), 65 (C5H5+, 11.6), 39

Experimental

101

(C3H3+, 10.8). IR (ATR) [cm-1] = 3032 (w), 2908 (w), 1637 (s), 1533 (m), 1494 (w), 1452

(w), 1440 (w), 1411 (w), 1373 (w), 1350 (w), 1300 (w), 1251 (w), 1228 (w), 1199 (w), 1153

(w), 1411 (w), 1373 (w), 1350 (w), 1300 (w), 1251 (w), 1228 (w), 1199 (w), 1153 (w), 1068

(w), 1014 (w), 891 (w), 771 (w), 702 (s), 684 (m), 665 (w), 638 (s). Anal. calcd. for

C12H13NOS (219.3): C 65.72, H 5.97, N 6.39. Found: C 65.89, H 6.11, N 6.30.

7.4.10. Methyl 3-(4-(2-oxo-2-(prop-2-yn-1-ylamino)ethoxy)phenyl)propanoate (3k)

220 mg (0.80 mmol, 80 %) of 3k was obtained as colorless crystalline solid after purification

by silica gel column chromatography (n-hexane:ethylacetate (3:1)).

Mp. 89 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.26 (t, 4J = 2.5 Hz, 1 H), 2.59 (t, 3J = 7.6 Hz,

2 H), 2.90 (t, 3J = 7.8 Hz, 2 H), 3.66 (s, 3 H), 4.14 (dd, 3J = 2.5, 5.5 Hz, 2 H), 4.48 (s, 2 H),

6.81 (br s, 1 H), 6.84 (d, 3J = 8.6 Hz, 2 H), 7.15 (d, 3J = 8.5 Hz, 2 H). 13C-NMR (75 MHz,

CDCl3): δ = 28.8 (CH2), 30.1 (CH2), 35.9 (CH2), 51.7 (CH3), 67.5 (CH2), 72.0 (CH),

79.1 (Cquat), 114.9 (CH), 129.7 (CH), 134.5 (Cquat), 155.7 (Cquat), 168.1 (Cquat), 173.4 (Cquat).

EI-MS: m/z (%) = 276 ([M + H]+, 6.4), 275 (M+, 34.6), 215 (6.6), 202 ([M - C3H5O2]+, 38.3),

133 (10.7), 137 (59.3), 138 (5.3), 193 (6.6), 179 ([M - C5H6NO]+, 100), 163 (5.3), 121

(C8H9O+, 14.6), 107 (C7H7O

+, 33.8), 105 (5.9), 103 (8.5), 96 (C5H6NO+, 20.7), 90 (6.0), 77

(C6H5+, 8.0), 55 (C3H5N

+, 7.9), 39 (C3H3+, 12.03). IR (ATR) [cm-1] = 2997 (w), 2953 (w),

2935 (w), 2918 (w), 2866 (w), 1720 (m), 1658. (s), 1624 (w), 1608 (w), 1587 (w), 1570 (w),

1529 (m), 1508 (s), 1448 (m), 1438 (m), 1417 (w), 1340 (m), 1300 (m), 1288 (m), 1232 (s),

1174 (m), 1141 (m), 1107 (m), 1093 (w), 1066 (m), 1024 (m), 993 (m), 925 (w), 875 (w), 856

(m), 840 (m), 821 (m), 808 (m), 731 (m), 698 (m), 657 (m). Anal. calcd. for C15H17NO4

(275.3): C 65.44, H 5.09, N 6.22. Found: C 65.37, H 5.06, N 6.32.

Experimental

102

7.4.11. (R)-N-(Prop-2-yn-1-yl)-2-(2,2,2-trifluoroacetimido)propanamide (3n)

138 mg (0.62 mmol, 62 %) of 3n was obtained as colorless solid after purification by silica

gel column chromatography (n-hexane:ethylacetate (3:1)).

Mp. 99 °C. 1H-NMR (300 MHz, CDCl3): δ = 1.48 (d, 3J = 6.9 Hz, 3 H), 2.26 (t, 4J = 2.6 Hz,

1 H), 4.06 (dd, 3J = 5.3 Hz, 4J = 2.6 Hz, 2 H), 4.56 (dq, 3J = 7.1 Hz, 7.1 Hz, 1 H), 6.46 (br s,

1 H), 7.41 (d, 3J = 5.4 Hz, 1 H). 13C-NMR (75 MHz, CDCl3): δ = 18.6 (CH3), 29.7 (CH2),

49.3 (CH), 72.4 (CH), 78.5 (Cquat), 115.7 (d, 1J = 287.2 Hz, Cquat), 157.1 (d, 2J = 37.9 Hz,

Cquat), 170.4 (Cquat). EI-MS: m/z (%) = 223 ([M + H]+, 0.4), 222 (M+, 0.5), 141 ([M - C4H3NO]+,

100), 140 ([M - C4H4NO]+, 31.5), 72 (12.3), 69 (8.9), 39 (C3H3+, 12.2). IR (ATR) [cm-1] =

3298 (w), 3093 (w), 1701 (m), 1660 (m), 1548 (m), 1452 (w), 1421 (w), 1381 (w), 1361 (w),

1344 (w), 1305 (w), 1265 (w), 1246 (w), 1182 (s), 1157 (s), 1062 (w), 1047 (w), 1006 (w),

929 (w), 894 (w), 840 (w), 786 (w), 607 (w), 702 (w), 659 (s). Anal. calcd. for C8H9F3N2O2

(222.2): C 43.25, H 4.08, N 12.61. Found: C 43.36, H 4.18, N 12.56.

7.4.12. (R)-Benzyl (1-oxo-1-(prop-2-yn-1-ylamino)propan-2-yl)carbamate (3o)

213 mg (0.82 mmol, 82 %) of 3o was obtained as colorless solid after purification by silica

gel column chromatography (n-hexane:ethylacetate (2:1)).

Experimental

103

Mp. 123 °C. 1H-NMR (300 MHz, d6-acetone): δ = 1.34 (d, 3J = 7.1 Hz, 3 H), 2.65 (t, 4J = 2.6

Hz, 1 H), 3.99 (dd, 3J = 5.6 Hz, 4J = 2.6 Hz, 2 H), 4.22 (m, 1 H), 5.06 (d, 4J = 3.2 Hz, 2 H),

6.52 (br s, 1 H), 7.27-7.38 (br m, 5 H), 7.62 (br s, 1 H). 13C-NMR (75 MHz, d6-DMSO): δ =

18.1 (CH3), 28.0 (CH2), 49.9 (CH), 65.4 (CH2), 73.0 (CH), 81.1 (Cquat), 127.8 (CH), 127.8

(CH), 128.3 (CH), 137.0 (Cquat), 155.7 (Cquat), 172.3 (Cquat). EI-MS: m/z (%) = 260 (M+, 1.0),

178 ([M - C4H4NO]+, 33.6), 153 (1.9), 134 ([M - C6H10N2O]+, 27.2), 91 (C7H7+, 100), 107 (3.2),

82 (3.0), 77 (C6H5+, 2.2), 65 (C5H5

+, 5.9), 39 (C3H3+, 6.8). IR (ATR) [cm-1] = 2980 (w), 2935

(w), 1681 (s), 1643 (s), 1529 (s), 1498 (w), 1498 (w), 1448 (w), 1386 (w), 1367 (w), 1328 (w),

1261 (m), 1232 (s), 1172 (w), 1111 (w), 1074 (w), 1058 (w), 1045 (m), 1029 (w), 1006 (w),

954 (w), 916 (w), 840 (w), 777 (w), 756 (w), 736 (w), 698 (m), 680 (m), 651 (m), 636 (m).

Anal. calcd. for C14H16N2O3 (260.3): C 64.60, H 6.20, N 10.76 Found: C 64.31, H 6.41,

N 10.55.

7.4.13. (S)-Benzyl (1-oxo-1-(prop-2-yn-1-ylamino)propan-2-yl)carbamate (3p)

208 mg (0.80 mmol, 80 %) of 3p was obtained as colorless solid after purification by silica

gel column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 123 °C. 1H-NMR (300 MHz, d6-acetone): δ = 1.33 (d, 3J = 7.1 Hz, 3 H), 2.64 (t, 4J = 2.6

Hz, 1 H), 3.99 (dd, 3J = 5.6, 4J = 2.6 Hz, 2 H), 4.21 (m, 1 H), 5.07 (d, 4J = 3.1 Hz, 2 H), 6.50

(br s, 1 H), 7.27-7.38 (m, 5 H), 7.60 (br s, 1 H). 13C-NMR (75 MHz, d6-acetone): δ = 18.7

(CH3), 29.0 (CH2), 51.3 (CH), 66.8 (CH2), 72.0 (CH), 81.2 (Cquat), 128.6 (CH), 129.2 (CH),

138.2 (Cquat), 156.7 (Cquat), 172.8 (Cquat). EI-MS: m/z (%) = 261 ([M + H]+, 0.8), 260 (M+, 1.3),

178 ([M - C4H4NO]+, 35.9), 153 ([M - C7H7O]+, 2.6), 146 (4.1), 134 (C8H6O2+, 28.9), 107 (3.0),

91 (C7H7+, 100), 88 (8.2), 82 (2.9), 77 (C6H5

+, 2.1), 65 (C5H5+, 6.9), 39 (C3H3

+, 7.4). IR (ATR)

[cm-1] = 3037 (w), 2976 (w), 2929 (w), 2781 (w), 2771 (w), 1681 (s), 1643 (s), 1529 (s),

1498 (w), 1448 (w), 1415 (w), 1366 (w), 1367 (w), 1328 (w), 1259 (s), 1232 (s), 1172 (w),

1111 (w), 1074 (w), 1056 (m), 1045 (m), 1030 (w), 1008 (w), 956 (w), 916 (w), 840 (w), 777

Experimental

104

(w), 756 (w), 736 (w), 698 (s), 678 (s), 651 (s), 636 (s), 621 (w). Anal. calcd. for C14H16N2O3

(260.3): C 64.40, H 6.20, N, 10.76. Found: C 64.40, H 6.02, N 10.63.

7.4.14. (S)-Benzyl 2-(prop-2-yn-1-ylcarbamoyl)pyrrolidine-1-carboxylate (3q)

195 mg (0.68 mmol, 68 %) of 3q was obtained as colorless crystalline solid after purification

by silica gel column chromatography (n-hexane:ethylacetate (1:1)).

Mp. 96 °C. 1H-NMR (300 MHz, d6-acetone): δ = 1.90-1.94 (m, 2 H), 1.98-2.01 (m, 1 H), 2.12-

2.22 (m, 1 H), 2.64 (t, 4J = 2.6 Hz, 1 H), 3.46-3.54 (m, 2 H), 3.98 (dd, 3J = 5.5 Hz, 4J = 2.3

Hz, 2 H), 4.28 (dd, 3J = 8.3 Hz, 4J = 3.3 Hz, 1 H), 5.02-5.18 (m, 2 H), 7.34-7.39 (m, 5H), 7.63

(br s, 1 H). 13C-NMR (75 MHz, d6-acetone): δ = 24.1 (CH2), 28.9 (CH2), 32.0 (CH2), 47.6

(CH2), 61.5 (CH), 67.0 (CH2), 71.9 (CH), 81.4 (Cquat), 128.3 (CH), 128.5 (CH), 129.2

(CH), 138.2 (Cquat), 155.9 (Cquat), 172.8 (Cquat). EI-MS: m/z (%) = 286 (M+, 1.8), 204 ([M -

C4H4NO]+, 40.1), 178 (3.2), 161 (3.8), 160 (37.0), 151 ([M - C8H7O2]+, 3.1), 134 (3.6), 111

(1.2), 105 (2.9), 91 (C7H7+, 100), 77 (C6H5

+, 2.2), 65 (C5H5+, 5.8), 57 (2.6), 39 (C3H3

+, 5.1),

41 (3.5). IR (ATR) [cm-1] = 3062 (w), 2970 (w), 2947 (w), 2929 (w), 2872 (w), 2773 (w),

2459 (w), 2125 (w), 1747 (w), 1707 (s), 1651 (s), 1606.70 (w), 1541 (w), 1496 (w), 1485 (w),

1458 (w), 1458 (w), 1446 (w), 1415 (s), 1357 (s), 1309 (w), 1278 (w), 1236 (w), 1205 (w),

1186 (w), 1170 (w), 1126 (m), 1091 (w), 1076 (w), 1028 (w), 1014 (w), 987 (w), 968 (w), 925

(w), 900 (w), 887 (w), 802 (w), 769 (w), 752 (w), 729 (s), 694 (m), 680 (m), 621 (w), 605 (w).

Anal. calcd. for C16H18N2O3 (286.3): C 67.12, H 6.34, N 9.78. Found: C 67.00, H 6.15,

N 9.62.

Experimental

105

7.4.15. Benzyl (3-hydroxy-1-oxo-1-(prop-2-yn-1-ylamino)propan-2-yl)carbamate (3r)

146 mg (0.53 mmol, 53 %) of 3r was obtained as colorless crystalline solid after purification

by silica gel column chromatography (elution started with n-hexane:ethylacetate 1:1, product

obtainment with n-hexane:ethylacetate (1:4)).

Mp. 128 °C. 1H-NMR (300 MHz, d6-acetone): δ = 2.64 (t, 4J = 2.6 Hz, 1 H), 3.74-3.88 (m,

2 H), 4.00 (dd, 3J = 5.5 Hz, 4J = 2.5 Hz, 2 H), 4.16-4.27 (m, 2 H), 5.09 (d, 4J = 1.4 Hz, 2 H),

6.39 (br s, 1 H), 7.28-7.435 (m, 5 H), 7.70 (br s, 1 H). 13C-NMR (75 MHz, d6-DMSO): δ = 28.1

(CH2), 57.1 (CH), 61.7 (CH2), 65.5 (CH2), 73.0 (CH), 81.1 (Cquat), 127.8 (CH), 127.8 (CH),

128.4 (CH), 137.0 (Cquat), 155.9 (Cquat), 169.9 (Cquat). EI-MS: m/z (%) = 276 (M+, 0.7), 258 ([M

- H2O]+, 2.6), 194 ([M - C4H4NO]+, 24.5), 177 (2.1), 169 ([M - C7H7O]+, 2.9), 150 ([M -

C6H8NO2]+, 20.9), 151 (C8H9NO2

+, 2.3), 91 (C7H7+, 100). IR (ATR) [cm-1] = 3336 (w),

3273 (m), 3265 (w), 3057 (w), 2980 (w), 2970 (w), 2931 (w), 2860 (w), 2808 (w), 1701 (s),

1670 (s), 1544 (s), 1492 (w), 1458 (m), 1419 (w), 1392 (w), 1328 (w), 1284 (m), 1265 (w),

1244 (m), 1234 (m), 1211 (m), 1118 (w), 1093 (m), 1070 (m), 1058 (m), 1039 (m), 1012 (m),

987 (w), 939 (w), 925 (w), 825 (w), 785 (w), 750 (s), 663 (s), 615 (w). Anal. calcd. for

C14H16N2O4 (276.3): C 60.86, H 5.84, N 10.14. Found: C 60.75, H 5.98, N 10.01.

7.4.16. N-(Prop-2-yn-1-yl)furan-2-carboxamide (3s)

106 mg (0.71 mmol, 71 %) of 3s was obtained as colorless crystalline solid after purification

by silica gel column chromatography (n-hexane:ethylacetate (2:1)).

Experimental

106

Mp. 81 °C. 1H NMR (300 MHz, CDCl3): δ = 2.27 (t, 4J = 2.6 Hz, 1 H), 4.22 (dd, 3J = 2.6 Hz,

5.5 Hz, 2 H), 6.49 (dd, 3J = 1.8 Hz, 3.5 Hz, 1 H), 6.58 (br s, 1 H), 7.14 (d, 3J = 3.5 Hz, 1 H),

7.44 (d, 3J = 1.5 Hz, 1 H). 13C-NMR (75 MHz, CDCl3): δ = 28.9 (CH2), 71.9 (CH), 79.4 (Cquat),

112.3 (CH), 114.9 (CH), 144.3 (CH), 147.5 (Cquat), 158.0 (Cquat). EI-MS: m/z (%) = 150

([M + H]+, 4.4), 149 (M+, 21.9), 148 (2.3), 121 ([M - C2H4]+, 21.2), 106 (5.8), 95 ([M - C3H4N]+,

100), 93 (C5HO2+, 40.8), 78 (7.1), 67 ([M - C4H4NO]+, 10.8), 52 (5.4), 39 (C3H3

+, 31.7). IR

(ATR) [cm-1] = 3263 (w), 3180 (w), 1653 (m), 1637 (s), 1589 (m), 1571 (w), 1525 (m), 1473

(m), 1436 (w), 1415 (m), 1381 (w), 1348 (w), 1300 (m), 1265 (w), 1255 (w), 1230 (w), 1186

(m), 1147 (w), 1082 (w), 1018 (m), 923 (w), 908 (w), 885 (w), 835 (w), 786 (w), 756 (s), 721

(m), 671 (m), 636 (s), 613 (s). Anal. calcd. for C8H7NO2 (149.1): C 64.43, H 9.39, N 4.73.

Found: C 64.21, H 9.36, N 4.59.

7.4.17. N-(Prop-2-yn-1-yl)thiophene-2-carboxamide (3t)

127 mg (0.77 mmol, 77 %) of 3t was obtained as colorless crystalline solid after purification

by silica gel column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 116 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.27 (t, 4J = 2.6 Hz, 1 H), 4.22 (dd, 3J = 5.3 Hz,

4J = 2.6 Hz, 2 H), 6.41 (br s, 1 H), 7.07 (dd, 3J = 5.0 Hz, 3.8 Hz, 1 H), 7.49 (dd, 3J = 4.9 Hz,

4J = 1.1 Hz, 1 H), 7.56 (dd, 3J = 3.7 Hz, 4J = 1.2 Hz, 1 H). 13C-NMR (75 MHz, CDCl3): δ =

29.8 (CH2), 72.0 (CH), 79.5 (Cquat), 127.8 (CH), 128.7 (CH), 130.5 (CH), 138.2 (Cquat), 161.7

(Cquat). EI-MS: m/z (%) = 166 ([M + H]+, 2.0), 165 (M+, 15.7), 164 (7.3), 122 (9.5), 138 (3.0),

137 (8.2), 136 (54.8), 132 (5.3), 121 (5.1), 111 ([M -C3H4N]+, 100), 109 (4.5), 83 (C4H3S+,

10.0), 39 (C3H3+, 22.5). IR (ATR) [cm-1] = 3266 (w), 1625 (m), 1546 (s), 1514 (w), 1413

(w), 1357 (w), 1344 (w), 1305 (m), 1263 (w), 1246 (w), 1145 (w), 1078 (w), 1033. (w), 962

(w), 910 (w), 846 (w), 788 (w), 752 (w), 711 (m), 663 (m), 632 (s). Anal. calcd. for C8H7NOS

(165.2): C 58.16, H 4.27, N 8.48. Found: C 58.20, H 4.13, N 8.37.

Experimental

107

7.4.18. 2-(Piperidine-1-yl)-N-(prop-2-yn-1-yl)acetamide (3u)

126 mg (0.70 mmol, 70 %) of 3u was obtained as brown solid after purification by silica gel

column chromatography (n-hexane:ethylacetate (2:1)).

Mp. 62 °C. 1H-NMR (300 MHz, CDCl3): δ = 1.43 (m, 2 H), 1.58 (m, 4 H), 2.21 (t , 4J = 2.6 Hz,

1 H), 2.44 (t, 3J = 5.4 Hz, 4 H), 2.95 (s, 2 H), 4.06 (dd, 3J = 5.6 Hz, 4J = 2.6 Hz, 2 H), 7.43 (br

s, 1 H). 13C-NMR (75 MHz, CDCl3): δ = 23.8 (CH2), 26.3 (CH2), 28.7 (CH2), 55.1 (CH2), 62.3

(CH2), 71.3 (CH), 79.9 (Cquat), 170.8 (Cquat). EI-MS: m/z (%) = 180 (M+, 0.2), 98 ([M -

C4H4NO]+, 100), 84 ([M - C5H6NO]+, 6.4), 69 (C5H9+, 5.3), 55 (C3H5N

+, 16.2). IR (ATR)

[cm-1] = 3336 (w), 2935 (m), 2912 (w), 2852 (w), 2810 (w), 2758 (w), 2117 (w), 1654 (s),

1622 (w), 1514 (s), 1506 (w), 1492 (w), 1454 (w), 1415 (m), 1384 (w), 1367 (w), 1350 (w),

1332 (m), 1298 (w), 1273 (m), 1257 (m), 1161 (w), 1130 (m), 1109 (m), 1085 (m), 1039 (m),

1020 (w), 1001 (m), 979 (m), 960 (w), 931 (w), 866 (w), 852 (w), 792 (w), 731 (s), 702 (s),

671 (m), 611 (m). Anal. calcd. for C10H16N2O (180.2): C 66.63, H 8.95, N 15.54. Found:

C 66.44, H 8.73, N 15.38.

7.4.19. 3-Phenyl-N-(prop-2-yn-1-yl)propiolamide (3aa)

147 mg (0.80 mmol, 80 %) of 3aa was obtained as colorless crystalline solid after purification

by silica gel column chromatography (n-hexane:ethylacetate (3:1)).

Mp. 62 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.29 (t, 4J = 2.6 Hz, 1 H), 4.14 (dd, 3J = 2.6 Hz,

4J = 5.4 Hz, 2 H), 6.11 (br s, 1 H), 7.33-7.46 (m, 3 H), 7.52-7.60 (m, 2 H). 13C-NMR (75 MHz,

CDCl3): δ = 29.7 (CH2), 72.4 (CH), 78.6 (Cquat), 82.5 (Cquat), 85.8 (Cquat), 120.1 (Cquat), 128.7

(CH), 130.4 (CH), 132.7 (CH), 153.1 (Cquat). EI-MS: m/z (%) = 184 ([M + H]+, 4.0), 183 (M+,

Experimental

108

17.5), 182 ([M - H]+, 13.3), 154 (37.5), 155 (12.2), 141 (3.8), 140 (25.9), 129 ([M - C3H4N]+,

100), 130 (9.7), 139 (16.4), 114 (2.2), 126 (31.1), 101 (C8H5+, 11.1), 102 (C8H6

+, 13.8), 74

(5.2), 77 (C6H5+, 4.8), 75 (C6H3

+, 16.0), 63 (2.3), 52 (2.2), 51 (6.8), 39 (C3H3+, 3.1). IR (ATR)

[cm-1] = 3047 (w), 2667 (w), 2501 (w), 2216 (w), 2088 (w), 1749 (m), 1697 (m), 1683 (m),

1624 (m), 1595 (w), 1541 (s), 1521 (m), 1489 (w), 1338 (w), 1303 (m), 1288 (m), 1219 (w),

925 (w), 756 (w), 684 (m), 657 (m), 636 (m), 609 (w). Anal. calcd. for C12H9NO (183.2): C

78.67, H 4.95, N 7.65. Found: C 78.54, H 5.12, N 7.59.

7.5. Procedure for Chemoenzymatic One-pot Synthesis of Amide ligated

1,4-Disubstituted 1,2,3-Triazoles 5

To a 2.0 mL MTBE solution of 55 mg (1.00 mmol) of propargylamine (2), in a screwcap

Schlenk vessel, were added 1.20 mmol of the ester 1 (experimental details mentioned in

Table 7.2) and Novozyme® 435 (50 % w/w of corresponding ester substrate 1 used) and the

reaction was shaken in an incubating shaker at 45 °C for 4-24 h (depending upon the nature

of ester 1 used). After the completion of aminolysis, 1.50 mmol of the azide 4a-b, 6.0 mg

(0.04 mmol) of Cu2O, 9.7 mg (0.08 mmol) of benzoic acid, and 1.0 mL of water, were added

to the reaction vessel and the reaction mixture was shaken at 45 °C for 4 h. It is important to

mention that methanol as co solvent was added only if the amide product 3 of enzymatic

step was insoluble in MTBE and crystallized out of the reaction mixture during the course of

the reaction. The reaction mixture containing colorless precipitated triazole product 5 was

dissolved in ethylacetate (10.0 mL) to filter the enzyme beads. To this diluted filtered

reaction mixture was added brine (5.0 mL) followed by extraction with ethylacetate (3 x 10.0

mL). The combined organic layers were dried with anhydrous Na2SO4 and the crude product

was purified by flash chromatography on silica gel using n-hexane/ethylacetate as eluent, to

get the analytically pure triazoles 5.

Experimental

109

Table 7.2. Experimental details of chemoenzymatic one-pot synthesis of 1,4-disubstituted

1,2,3-triazoles 5 using 1.50 mmol (200 mg) of benzylazide (4a).

Entry Ester Substrate 1 Triazole 5

1 233 mg (1.20 mmol) of 1a 249 mg (71 %) of 5aa

2 197 mg (1.20 mmol) of 1b 195 mg (61 %) of 5ba

3 199 mg (1.20 mmol) of 1e 268 mg (83 %) of 5eb

4 216 mg (1.20 mmol) of 1f 235 mg (70 %) of 5fb

5 198 mg (1.20 mmol) of 1h 273 mg (85 %) of 5hb

6 219 mg (1.20 mmol) of 1i 250 mg (74 %) of 5ib

7 236 mg (1.20 mmol) of 1j 233 mg (66 %) of 5jb

8 303 mg (1.20 mmol) of 1k 208 mg (51 %) of 5kb

9 239 mg (1.20 mmol) of 1n 210 mg (59 %) of 5nb

10 285 mg (1.20 mmol) of 1o 275 mg (70 %) of 5ob

11 285 mg (1.20 mmol) of 1p 287 mg (73 %) of 5pb

12 151 mg (1.20 mmol) of 1s 192 mg (68 %) of 5sa

13 171 mg (1.20 mmol) of 1t 104 mg (35 %) of 5ta

14 192 mg (1.20 mmol) of 1aa 225 mg (71 %) of 5aaa

15 204 mg (1.20 mmol) of 1dd 103 mg (40 %) of 5ddb

16 222 mg (1.20 mmol) of 1ee 208 mg (61 %) of 5eea

aReaction time of 24 h for Novozyme

® 435 catalyzed aminolysis.

bReaction time of 4 h for Novozyme

®

435 catalyzed aminolysis.

Table 7.3. Experimental details of chemoenzymatic one-pot synthesis of 1,4-disubstituted

1,2,3-triazoles 5 using 1.50 mmol (248 mg) of azidomethyl phenyl sulphide (4b).

Entry Ester Substrate 1 Triazole 5

1 197 mg (1.20 mmol) of 1b 222 mg (63 %) of 5ffa

2 198 mg (1.20 mmol) of 1h 276 mg (78 %) of 5ggb

3 219 mg (1.20 mmol) of 1i 218 mg (59 %) of 5hhb

4 151 mg (1.20 mmol) of 1s 267 mg (85 %) of 5iia

aReaction time of 24 h for Novozyme

® 435 catalyzed aminolysis.

bReaction time of 4 h for Novozyme

®

435 catalyzed aminolysis.

Experimental

110

7.6. Analytical Data of Amide Ligated 1,4-Disubstituted 1,2,3-Triazoles (5)

7.6.1. N-((1-Benzyl-1H- 1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)propanamide

(5a)

249 mg (0.71 mmol, 71 %) of 5a was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:20)).

Mp. 128 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 2.35 (t, 3J = 8.0 Hz, 2 H), 2.74 (t, 3J = 8.0

Hz, 2 H), 3.70 (s, 3 H), 4.26 (d, 3J = 5.6 Hz, 2 H), 5.55 (s, 2 H), 6.80 (d, 3J = 8.6 Hz, 2 H),

7.09 (d, 3J = 8.6 Hz, 2 H), 7.30-7.39 (m, 5 H), 7.83 (s, 1 H), 8.30 (t, 3J = 5.5 Hz, 1 H). 13C-

NMR (75 MHz, d6-DMSO): δ = 30.1 (CH2), 34.1 (CH2), 37.1 (CH2), 52.7 (CH3), 54.9 (CH2),

113.7 (CH), 122.8 (CH), 127.9 (CH), 128.1 (CH), 128.7 (CH), 129.1 (CH), 133.1 (Cquat),

136.1 (Cquat), 145.3 (Cquat), 157.5 (Cquat), 171.3 (Cquat). EI-MS: m/z (%) = 351 ([M + H]+ , 22.8),

350 (M+, 92.9), 189 (C10H13N4+, 10.7), 187 (C10H11N4

+, 21.7), 173 (C10H11N3+, 45.2), 159

(5.2), 144 (10.7), 135 (17.3), 134 (C9H10O+, 100), 121 (C8H9O

+, 63.4), 97 (8.5), 91 (C7H7+,

77.8), 86 (4.0), 77 (C6H5+, 7.5), 69 (4.1), 65 (C5H5

+, 8.1). IR (ATR) [cm-1] = 3010 (w), 2960

(w), 2880 (w), 1639 (m), 1612 (w), 1548 (m), 1512 (m), 1492 (w), 1438 (w), 1425 (w),

1367 (w), 1288 (w), 1249 (m), 1220 (m), 1174 (w), 1107 (w), 1074 (w), 1055 (m), 1029 (m),

1002 (w), 817 (m), 788 (w), 761 (w), 719 (s), 694 (m), 669 (w). Anal. calcd. for C20H22N4O2

(350.4): C 68.55, H 6.33, N 15.99. Found: C 68.49, H 6.14, N 15.87.

Experimental

111

7.6.2. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-phenylpropanamide (5b)

195 mg (0.61 mmol, 61 %) of 5b was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1: 20)).

Mp. 121 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 2.39 (t, 3J = 7.7 Hz, 2 H), 2.81 (t, 3J = 7.7

Hz, 2 H), 4.27 (d, 3J = 5.6 Hz, 2 H), 5.55 (s, 2 H), 7.12-7.40 (br m, 10 H), 7.80 (s, 1 H), 8.32

(t, 3J = 5.3 Hz, 1 H). 13C-NMR (75 MHz, d6-DMSO): δ = 31.0 (CH2), 34.1 (CH2), 36.8 (CH2),

52.7 (CH2), 122.8 (CH), 125.9 (CH), 127.9 (CH), 128.1 (CH), 128.2 (CH), 128.2 (CH), 128.7

(CH), 136.1 (Cquat), 141.3 (Cquat), 145.0 (Cquat), 171.2 (Cquat). EI-MS: m/z (%) = 322 ([M +

2H]+, 1.3), 321 ([M + H]+

, 13.1), 320 (M+, 59.0), 211 (3.4), 187 ([M - C9H9O]+, 26.0), 173

(C10H11N3+, 36.7), 159 (C9H9N3

+, 9.2), 144 (13.8), 143 (21.7), 132 (C7H6N3+, 4.0), 133 (2.3),

105 (C8H9+, 14.7), 91 (C7H7

+, 100), 77 (C6H5+, 4.8), 65 (C5H5

+, 8.9), 39 (C3H3+, 2.3). IR (ATR)

[cm-1] = 3030 (w), 2929 (w), 2912 (w), 1637 (m), 1604 (w), 1546 (m), 1492 (w), 1454 (w),

1438 (w), 1381 (w), 1365 (w), 1336 (w), 1321 (w), 1301 (w), 1290 (w), 1259 (w), 1002 (w),

1220 (w), 1174 (w), 1163 (w), 1126 (w), 1076 (w), 1055 (w), 1028 (w), 860 (w), 842 (w), 827

(w), 785 (w), 761 (w), 717 (s), 694 (w), 669 (w), 651 (w), 621 (w). Anal. calcd. for C19H20N4O

(320.4) : C 71.23, H 6.29, N 17.49. Found: C 71.20, H 6.36, N 17.47.

Experimental

112

7.6.3. N-((1-Benzyl-1H-1,2,3-triazole-4-yl)methyl)-2-phenoxyacetamide (5e)

268 mg (0.83 mmol, 83 %) of 5e was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:10)).

Mp. 129 °C. 1H-NMR (300 MHz, CDCl3): δ = 4.44 (d, 3J = 6.0 Hz, 2 H), 4.55 (s, 2 H), 5.45 (s,

2 H), 6.84 (d, 3J = 8.7 Hz, 2 H), 6.96 (t, 3J = 7.4 Hz, 1 H), 7.20 (br s, 1 H), 7.21-7.25 (m, 4 H),

7.32-7.36 (m, 3 H), 7.39 (s, 1 H). 13C-NMR (75 MHz, CDCl3): δ = 34.5 (CH2), 54.4 (CH2),

67.3 (CH2), 114.7 (CH), 122.2 (CH), 128.3 (CH), 129.0 (CH), 129.3 (CH), 129.9 (CH), 134.5

(Cquat), 144.8 (Cquat), 157.2 (Cquat), 168.4 (Cquat). EI-MS: m/z (%) = 322 (M+, 4.4), 231 ([M -

C7H7]+, 1.6), 229 ([M - C6H5O]+, 5.9), 215 ([M - C7H7O]+, 6.0), 174 (9.2), 173 (C10H11N3

+,

72.8), 187 (6.6), 107 (8.7), 91 (C7H7+, 100). IR (ATR) [cm-1] = 2939 (w), 2908 (w), 2852

(w), 1656 (m), 1600 (w), 1539 (w), 1492 (w), 1438 (w), 1427 (w), 1291 (w), 1029 (w),

1226 (m), 1176 (w), 1130 (w), 1083 (w), 1053 (m), 855 (w), 833 (w), 798 (w), 748 (s), 719

(m), 692 (s), 669 (w). Anal. calcd. for C18H18N4O2 (322.4): C 67.07, H 5.63, N 17.38. Found:

C 66.93, H 5.68, N 17.30.

Experimental

113

7.6.4. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(benzyloxy)acetamide (5f)

235 mg (0.70 mmol, 70 %) of 5f was obtained as colorless solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:10)).

Mp. 100 °C. 1H-NMR (300 MHz, CDCl3): δ = 3.96 (s, 2 H), 4.52 (s, 2 H), 4.54 (s, 2 H), 5.49

(s, 2 H), 7.14 (br s, 1 H), 7.27-7.39 (m, 10 H), 7.43 (s, 1 H). 13C-NMR (75 MHz, CDCl3): δ =

34.4 (CH2), 54.3 (CH2), 69.5 (CH2), 73.7 (CH2), 122.2 (CH), 128.1 (CH), 128.3 (CH), 128.4

(CH), 128.7 (CH), 128.9 (CH), 129.3 (CH), 134.6 (Cquat), 136.8 (Cquat), 145.0 (Cquat), 169.7

(Cquat). EI-MS: m/z (%) = 336 (M+, 0.1), 245 (0.2), 230 (C12H14N4O+, 13.6), 187 (5.0), 91

(C7H7+, 100), 77 (C6H5

+, 2.6), 65 (C5H5+, 16.0). IR (ATR) [cm-1] = 3062 (w), 2999 (w),

2953 (w), 1654 (m), 1517 (w), 1494 (w), 1452 (w), 1373 (w), 1344 (w), 1311 (w), 1242 (w),

1226 (w), 1207 (w), 1132 (w), 1116 (m), 1074 (w), 1058 (m), 1029 (w), 1010 (w), 958 (w),

889 (w), 827 (w), 798 (w), 777 (w), 732 (m), 723 (s), 694 (s), 673 (w), 644 (w), 615 (w). Anal.

calcd. for C19H20N4O2 (336.4): C 67.84, H 5.99, N 16.66. Found: C 68.04, H 6.14, N 16.78.

Experimental

114

7.6.5. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(phenylamino)acetamide (5h)

273 mg (0.85 mmol, 85 %) of 5h was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:20)).

Mp. 116 °C. 1H-NMR (300 MHz, CDCl3): δ = 3.78 (br s, 2 H), 4.31 (br s, 1 H), 4.50 (d, 3J =

5.4 Hz, 2 H), 5.44 (s, 2 H), 6.54 (d, 3J = 7.3 Hz, 2 H), 6.77 (t, 3J = 6.7 Hz, 1 H), 7.14 (m, 2 H),

7.24 (m, 2 H), 7.36 (br m, 5 H). 13C-NMR (75 MHz, CDCl3): δ = 34.8 (CH2), 48.8 (CH2), 54.3

(CH2), 113.3 (CH), 119.2 (CH), 122.1 (CH), 128.3 (CH), 128.9 (CH), 129.3 (CH), 129.5 (CH),

134.6 (Cquat), 145.2 (Cquat), 147.1 (Cquat), 170.9 (Cquat). EI-MS: m/z (%) = 323 ([M + 2H]+, 1.5),

322 ([M + H]+, 11.7), 321 (M+, 52.1), 215 ([M - C7H8N]+, 3.2), 189 (C10H13N4+, 94.2), 173

(C10H11N3+, 67.0), 159 (C9H9N3

+, 4.5), 144 (C8H6N3+, 29.5), 106 (C7H8N

+, 100), 91 (C7H7+,

95.0), 77 (C6H5+, 21.7), 65 (C5H5

+, 8.2), 39 (C3H3+, 3.1). IR (ATR) [cm-1] = 3334 (w),

3032 (w), 2879 (w), 2831 (w), 1633 (s), 1602 (m), 1516 (m), 1494 (m), 1456 (w), 1429 (w),

1363 (w), 1313 (m), 1274 (w), 1261 (w), 1242 (w), 1228 (w), 1207 (w), 1180 (w), 1134 (w),

1118 (w), 1105 (w), 1074 (w), 1053 (m), 1026 (w), 991 (w), 858 (w), 796 (w), 767 (w), 748

(m), 725 (s), 686 (s), 663 (w), 638 (w), 617 (w). Anal. calcd. for C18H19N5O (321.4): C 67.27,

H 5.96, N 21.79. Found: C 67.00, H 5.89, N 21.72.

Experimental

115

7.6.6. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(phenylthio)acetamide (5i)

250 mg (0.74 mmol, 74 %) of 5i was obtained as colorless solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:10)).

Mp. 112 °C. 1H-NMR (300 MHz, CDCl3): δ = 3.62 (s, 2 H), 4.48 (d, 3J = 5.9 Hz, 2 H), 5.43 (s,

2 H), 7.13-7.24 (m, 8 H), 7.33-7.38 (m, 4 H). 13C-NMR (75 MHz, CDCl3): δ = 35.4 (CH2), 37.5

(CH2), 54.3 (CH2), 122.0 (CH), 126.8 (CH), 128.2 (CH), 128.5 (CH), 128.9 (CH), 129.3 (CH),

129.3 (CH), 134.6 (Cquat), 144.9 (Cquat), 168.1 (Cquat). EI-MS: m/z (%) = 339 ([M + H]+, 4.3),

338 (M+, 16.0), 320 (2.2), 305 (2.0), 229 (1.3), 215 (5.8), 189 (C10H13N4+, 86.7), 187 (5.5),

172 (11.2), 159 (2.2), 150 (4.3), 144 (C8H6N3+, 31.7), 123 (12.5), 109 (4.6), 91 (C7H7

+, 100),

77 (C6H5+, 6.2), 65 (C5H5

+, 10.9), 39 (C3H3+, 3.6). IR (ATR) [cm-1] = 3059 (w), 2985 (w),

2951 (w), 2908 (w), 1651 (s), 1622 (w), 1516 (m), 1479 (w), 1454 (w), 1438 (w), 1427 (w),

1404 (w), 1372 (w), 1300 (w), 1244 (w), 1199 (w), 1184 (w), 1168 (w), 1128 (w), 1091 (w),

1074 (w), 1055 (m), 1024 (w), 1002 (w), 896 (w), 871 (w), 788 (w), 769 (w), 734 (s), 719 (s),

688 (s), 661 (w), 613 (w). Anal. calcd. for C18H18N4OS (338.4): C 63.88, H 5.36, N 16.56.

Found: C 63.67, H 5.20, N 16.44.

Experimental

116

7.6.7. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(benzylthio)acetamide (5j)

233 mg (0.66 mmol, 66 %) of 5j was obtained as colorless solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:10)).

Mp. 107 °C. 1H-NMR (300 MHz, CDCl3): δ = 3.10 (s , 2 H), 3.65 (s , 2 H), 4.41 (d , 3J = 5.8

Hz, 2 H), 5.49 (s, 2 H), 7.17-7.24 (m, 6 H), 7.27-7.40 (m, 5 H), 7.42 (s, 1 H). 13C-NMR (75

MHz, CDCl3): δ = 35.2 (CH2), 35.3 (CH2), 37.1 (CH2), 54.3 (CH2), 122.2 (CH), 127.5 (CH),

128.2 (CH), 128.79 (CH), 129.0 (CH), 129.1 (CH), 129.3 (CH), 134.6 (Cquat), 137.1 (Cquat),

145.0 (Cquat), 168.8 (Cquat). EI-MS: m/z (%) = 353 ([M + H]+, 0.4), 352 (M+, 0.4), 261 ([M -

C7H7]+, 2.2), 230 (C12H14N4O

+, 98.1), 212 (1.2), 187 ([M - C9H9OS]+, 18.6), 172 ([M -

C9H10NOS]+, 15.1), 159 (C9H9N3+, 2.0), 144 (C8H6N3

+, 9.4), 139 (C8H11S+, 7.3), 115 (2.1), 111

(9.8), 97 (C4H5N2O+, 8.6), 91 (C7H7

+, 100), 77 (C6H5+, 1.7), 65 (C5H5

+, 9.3), 39 (C3H3+, 2.4).

IR (ATR) [cm-1] = 3034 (w), 2964 (w), 1668 (s), 1635 (w), 1558 (w), 1541 (w), 1494 (w),

1446 (w), 1408 (w), 1325 (w), 1303 (w), 1222 (m), 1213 (w), 1165 (w), 1149 (w), 1132 (w),

1056 (w), 1026 (w), 869 (w), 792 (w), 775 (w), 746 (w), 719 (m), 696 (s), 663 (w). Anal.

calcd. for C19H20N4OS (352.5): C 64.75, H 5.72, N 15.90. Found C 64.85, H 5.70, N 16.15.

Experimental

117

7.6.8. Methyl-3-(4-(2-(((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amino)-2-oxoethoxy)

phenyl)propanoate (5k)

208 mg (0.51 mmol, 51 %) of 5k was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1: 20)).

Mp. 106 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 2.57 (t, 3J = 7.4 Hz, 2 H), 2.78 (t, 3J = 7.5

Hz, 2 H), 3.57 (s, 3 H), 4.37 (d, 3J = 5.9 Hz, 2 H), 4.46 (s, 2 H), 5.56 (s, 2 H), 6.84 (d, 3J = 8.7

Hz, 2 H), 7.12 (d, 3J = 8.6 Hz, 2 H), 7.29-7.32 (m, 2 H), 7.34-7.40 (m, 3 H), 7.93 (s, 1 H),

8.59 (t, 3J = 5.8 Hz, 1 H). 13C-NMR (75 MHz, d6-DMSO): δ = 29.4 (CH2), 34.0 (CH2), 35.1

(CH2), 51.3 (CH3), 52.7 (CH2), 66.9 (CH2), 114.6 (CH2), 122.9 (CH), 128.0 (CH), 128.1 (CH),

128.7 (CH), 129.2 (CH), 133.1 (Cquat), 136.1 (Cquat), 145.1 (Cquat), 156.1 (Cquat), 167.8 (Cquat),

172.7 (Cquat). EI-MS: m/z (%) = 409 ([M + H]+, 4.3), 408 (M+, 16.7), 377 (6.8), 349 (1.8), 230

([M - C10H10O3]+, 45.2), 229 (9.6), 187 (9.0), 173 (C10H11N3

+, 40.1), 144 (C8H6N3+, 11.4), 91

(C7H7+, 100), 77 (C6H5

+, 21.7), 65 (C5H5+, 8.2), 39 (C3H3

+, 2.1). IR (ATR) [cm-1] = 3053 (w),

2954 (w), 2933 (w), 1730 (s), 1666 (s), 1612 (w), 1514 (s), 1494 (w), 1456 (w), 1435 (w),

1423 (w), 1375 (w), 1363 (w), 1332 (w), 1280 (w), 1244 (m), 1215 (w), 1203 (w), 1168 (m),

1118 (w), 1076 (w), 1053 (s), 1026 (w), 985 (w), 866 (w), 835 (w), 823 (s), 796 (w), 767 (w),

719 (s), 705 (s), 690 (w), 665 (w). Anal. calcd. for C22H24N4O4 (408.5): C 64.69, H 5.92,

N 13.72. Found: C 64.82, H 6.11, N 13.59.

Experimental

118

7.6.9. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)propan-

amide (5n)

210 mg (0.59 mmol, 59 %) of 5n was obtained as colorless solid after purification by column

chromatography (elution started with n-hexane:ethylacetate (1:1), product obtainment with n-

hexane:ethylacetate (1:20)).

Mp. 163 °C. 1H-NMR (300 MHz, d6-acetone): δ = 1.41 (d, 3J = 7.1 Hz , 3 H), 4.44 (d, 3J = 5.7

Hz, 2 H), 4.48-4.55 (m, 1 H), 5.59 (s, 2 H), 7.28-7.44 (m, 5 H), 7.82 (s, 1 H), 7.87 (br s, 1 H),

8.44 (br s, 1 H). 13C-NMR (75 MHz, d6-acetone): δ = 18.1 (CH3), 35.7 (CH2), 50.2 (CH), 54.1

(CH2), 117.0 (d, 1J = 287.4 Hz, Cquat), 123.2 (CH), 128.9 (CH), 129.1 (CH), 129.7 (CH), 137.1

(Cquat), 146.0 (Cquat), 157.1 (d, 2J = 36.83 Hz, Cquat), 171.4 (Cquat). EI-MS: m/z (%) = 356 ([M +

H]+, 3.1) , 355 (M+, 15.8), 215 ([M - C4H5F3NO]+, 9.0), 187 (C10H11N4+, 11.0), 173 (C10H11N3

+,

8.8), 149 (4.8), 143 (C8H5N3+, 32.0), 97 (C2F3O

+, 9.7), 91 (C7H7+, 100), 77 (C6H5

+, 6.9), 69

(CF3+, 69.2). IR (ATR) [cm-1] = 3078 (w), 3005 (w), 2951 (w), 2929 (w), 2879 (w), 1705 (m),

1656 (m), 1543 (m), 1496 (w), 1458 (w), 1348 (w), 1323 (w), 1213 (m), 1182 (s), 1155 (s),

1132 (w), 1107 (w), 1074 (w), 1053 (w), 1029 (w), 977 (w), 837 (w), 792 (w), 752 (w), 729

(m), 719 (m), 694 (m), 669 (w), 651 (w), 628 (w). Anal. calcd. for C15H16F3N5O2 (353.3): C

50.70, H 4.54, N 19.71 Found C 50.88, H 4.80, N 19.52.

Experimental

119

7.6.10. Benzyl 1-{[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amino}-1-oxopropan-2-yl

carbamate (5o)

275 mg (0.70 mmol, 70 %) of 5o was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1: 20)).

Mp. 166 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 1.19 (d, 3J = 7.2 Hz, 3 H), 4.02 (m, 1 H),

4.30 (d, 3J = 5.6 Hz, 2 H), 5.00 (d, 4J = 6.4, 2 H), 5.56 (s, 2 H), 7.29-7.39 (m, 10 H), 7.44 (d,

3J = 7.4 Hz, 1 H), 7.91 (s, 1 H), 8.36 (t, 3J = 5.5 Hz, 1 H). 13C-NMR (75 MHz, d6-DMSO): δ =

18.1 (CH3), 34.4 (CH2), 50.1 (CH), 52.7 (CH2), 65.4 (CH2), 122.8 (CH), 127.7 (CH), 127.8

(CH), 127.9 (CH), 128.1 (CH), 128.3 (CH), 128.7 (CH), 136.1 (Cquat), 137.0 (Cquat), 145.3

(Cquat), 155.7 (Cquat), 172.5 (Cquat). EI-MS: m/z (%): 393 (M+, 2.9), 350 (1.8), 286 (1.5), 244

(2.6), 216 (6.4), 221 (1.5), 215 (7.9), 189 (1.7), 188 (6.0), 174 (C10H12N3+, 10.0), 173

(C10H11N3+, 77.5), 143 (10.5), 144 (7.4), 117 (3.1), 107 (4.6), 108 (6.0), 91 (C7H7

+, 100), 79

(5.8), 65 (C5H5+, 7.0). IR (ATR) [cm-1] = 3062 (w), 2968 (w), 2954 (w), 1685 (m), 1639 (s),

1602 (w), 1587 (w), 1537 (m), 1496 (w), 1454 (w), 1454 (w), 1435 (w), 1392 (w), 1332 (w),

1305 (w), 1257 (m), 1232 (w), 1232 (w), 1220 (w), 1120 (w), 1078 (w), 1047 (m), 1029 (w),

848 (w), 779 (w), 758 (w), 717 (w), 698 (m), 671 (m), 646 (w). Anal. calcd. for C21H23N5O3

(393.4): C 64.11, H 5.89, N 17.80. Found: C 64.08, H 5.98, N 17.77.

Experimental

120

7.6.11. Benzyl-1-{[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amino}-1-oxopropan-2-yl

carbamate (5p)

287 mg (0.73 mmol, 73 %) of 5p was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1: 20)).

Mp. 166 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 1.19 (d, 3J = 7.1 Hz, 3 H), 4.00 (m, 1 H),

4.30 (d, 3J = 5.6 Hz, 2 H), 5.00 (d, 4J = 6.4 Hz, 2 H), 5.56 (s, 2 H), 7.29-7.39 (m, 10 H), 7.44

(d, 3J = 7.4 Hz, 1 H), 7.91 (s, 1 H), 8.37 (t, 3J = 5.5 Hz, 1 H). 13C-NMR (75 MHz, d6-DMSO):

δ = 18.1 (CH3), 34.4 (CH2), 50.1 (CH), 52.8 (CH2), 65.4 (CH2), 122.8 (CH), 127.7 (CH), 127.8

(CH), 128.0 (CH), 128.1 (CH), 128.4 (CH), 128.7 (CH), 136.1 (Cquat), 137.0 (Cquat), 145.3

(Cquat), 155.7 (Cquat), 172.6 (Cquat). EI-MS: m/z (%) = 394 ([M + H]+, 1.9), 393 (M+, 5.3), 350

(3.7), 286 (2.3), 215 (8.7), 188 (6.5), 189 (1.8), 173 (C10H11N3+ , 87.2), 174 (10.5), 159 (2.5),

143 (10.4), 144 (8.6), 130 (2.5), 108 (5.2), 91 (C7H7+, 100), 79 (4.9), 65 (C5H5

+, 6.9).

IR (ATR) [cm-1] = 3013 (w), 2913 (w), 2946 (w), 2906 (w), 2879 (w), 2853 (w), 2826 (w),

1685 (s), 1639 (s), 1616 (w), 1602 (w), 1587 (w), 1537 (s), 1496 (w), 1454 (w), 1435 (w),

1392 (w), 1332 (w), 1305 (w), 1257 (m), 1232 (m), 1220 (m), 1120 (w), 1078 (w), 1047 (w),

1028 (w), 848 (w), 821 (w), 779 (w), 758 (w), 734 (m), 717 (m), 698 (s), 671 (m), 646 (w),

619 (w). Anal. calcd. for C21H23N5O3 (393.4): C 64.11, H 5.89, N 17.80. Found: C 63.90, H

5.86, N 17.66.

Experimental

121

7.6.12. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)furan-2-carboxamide (5s)

192 mg (0.68 mmol, 68 %) of 5s was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1: 20)).

Mp. 94 °C. 1H-NMR (300 MHz, CDCl3): δ = 4.65 (d, 3J = 5.9 Hz, 2 H), 5.49 (s, 2 H), 6.46 (m,

1 H), 7.04 (br s, 1 H), 7.07 (dd, 3J = 3.5 Hz, 4J = 0.8 Hz, 1 H), 7.24 (m, 1 H), 7.28-7.38 (m, 4

H), 7.41 (m, 1 H), 7.51 (s, 1 H). 13C-NMR (75 MHz, CDCl3): δ = 34.7 (CH2), 54.4 (CH2), 112.2

(CH), 114.5 (CH), 122.4 (CH), 128.3 (CH), 128.9 (CH), 129.3 (CH), 134.5 (Cquat), 144.2

(CH), 145.0 (Cquat), 147.7 (Cquat), 158.5 (Cquat). EI-MS: m/z (%) = 283 ([M + H]+, 7.4), 282 (M+,

36.2), 254 ([M - N2]+, 5.0), 191 ([M - C7H7]

+, 11.7), 187 ([M - C5H3O2]+, 13.6), 159 (4.2), 144

(11.0), 95 (C5H3O2+, 48.7), 91 (C7H7

+, 100), 77 (C6H5+, 2.6), 65 (C5H5

+, 12.0), 39 (C3H3+,

10.6). IR (ATR) [cm-1] = 3037 (w), 2958 (w), 2924 (w), 2856 (w), 1639 (m), 1591 (m),

1575 (w), 1546 (w), 1525 (w), 1496 (w), 1469 (w), 1456 (w), 1423 (w), 1375 (w), 1328 (w),

1298 (w), 1284 (w), 1207 (w), 1192 (m), 1143 (w), 1111 (w), 1076 (w), 1041 (w), 1018 (w),

987 (w), 912 (w), 883 (w), 835 (w), 823 (w), 796 (w), 771 (w), 752 (s), 727 (w), 109 (s), 696

(s), 667 (w), 653 (w), 605 (m). Anal. calcd. for C15H14N4O2 (282.3): C 63.82, H 5.00, N 19.85.

Found: C 63.56, H 4.93, N 19.79.

Experimental

122

7.6.13. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)thiophene-2-carboxamide (5t)

104 mg (0.35 mmol, 35 %) of 5t was obtained as colorless solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1: 20)).

Mp. 123 °C. 1H-NMR (300 MHz, CDCl3): δ = 4.63 (d, 3J = 5.8 Hz, 2 H), 5.48 (s, 2 H), 7.02

(dd, 3J = 3.8 Hz, 4.9 Hz, 1 H), 7.25 (br s, 1 H), 7.27-7.39 (m, 5 H), 7.43 (dd, 3J = 5.0 Hz, 4J =

1.1 Hz, 1 H), 7.55 (dd, 3J = 3.7 Hz, 4J = 1.1 Hz, 1 H), 7.59 (s, 1 H). 13C-NMR (75 MHz,

CDCl3): δ = 35.7 (CH2), 54.8 (CH2), 123.3 (CH), 128.2 (CH), 128.7 (CH), 128.8 (CH), 129.4

(CH), 129.7 (CH), 130.7 (CH), 134.9 (Cquat), 139.2 (Cquat), 145.6 (Cquat), 162.6 (Cquat). EI-MS:

m/z (%) = 300 ([M + 2H]+, 2.6), 299 ([M + H]+, 7.5), 298 (M+, 38.6), 280 (3.1), 269 ([M - N2 -

H]+, 1.8), 207 ([M - C7H7]+, 3.7), 187 ([M - C5H3OS]+, 25.8), 179 (9.9), 173 (C10H11N3

+, 2.5),

159 (C9H9N3+, 8.2), 151 (1.9), 143 (C8H5N3

+, 39.0), 130 (8.2), 111 (C5H3OS+, 59.3), 104 (3.1),

91 (C7H7+, 100), 83 (C4H3S

+, 5.5), 77 (C6H5+, 2.4), 65 (C5H5

+, 12.7), 39 (C3H3+, 11.8). IR

(ATR) [cm-1] = 3080 (w), 3064 (w), 2927 (w), 2860 (w), 1620 (s), 1556 (s), 1514 (w), 1494

(w), 1456 (w), 1417 (m), 1377 (w), 1354 (m), 1327 (w), 1200 (s), 1263 (w), 1240 (m), 1228

(w), 1207 (w), 1145 (w), 1134 (w), 1111 (w), 1076 (w), 1047 (m), 1029 (w), 1018 (w), 952

(w), 860 (w), 819 (w), 794 (w), 752 (w), 725.23 (s), 709 (s), 694 (s), 665 (m), 653 (m), 619

(w). Anal. calcd. for C15H14N4OS (298.4): C 60.38, H 4.73, N 18.78. Found C 60.32, H 4.64,

N 18.60.

Experimental

123

7.6.14. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-phenylpropiolamide (5aa)

225 mg (0.71 mmol, 71 %) of 5aa was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (2:1), product

obtainment with n-hexane:ethylacetate (1:3)).

Mp. 137 °C. 1H-NMR (300 MHz, CDCl3): δ = 4.51 (d, 3J = 5.8 Hz, 2 H), 5.42 (s, 2 H), 6.91 (br

s, 1 H), 7.16-7.25 (m, 3 H), 7.28-7.35 (m, 5 H), 7.42-7.43 (m, 2 H), 7.45 (s, 1 H). 13C-NMR

(75 MHz, CDCl3): δ = 35.4 (CH2), 54.4 (CH2), 82.8 (Cquat), 85.4 (Cquat), 120.2 (Cquat), 122.5

(CH), 128.3 (CH), 128.6 (CH), 129.0 (CH), 129.3 (CH), 130.2 (CH), 132.7 (CH), 134.5

(Cquat), 144.4 (Cquat), 153.5 (Cquat). EI-MS: m/z (%) = 317 ([M + H]+, 4.5), 316 (M+, 18.5), 287

(6.8), 273 (4.2), 259 (3.7), 244 (4.2), 197 (11.5), 187 ([M - C9H5O]+, 16.8), 169 (3.8), 161

(4.4), 129 (C9H5O+, 58.3), 101 (4.8), 91 (C7H7

+, 100), 75 (6.2), 65 (C5H5+, 8.5), 39 (C3H3

+,

2.6). IR (ATR) [cm-1] = 3223 (w), 3143 (w), 3051 (w), 3034 (w), 2939 (w), 2862 (w),

2511 (w), 2229 (w), 1950 (w), 1730 (w), 1637 (s), 1552 (w), 1537 (w), 1489 (w), 1454 (w),

1159 (w), 1051 (w), 1421 (w), 1338 (w), 1300 (m), 1280 (m), 1238 (w), 1219 (s), 1122 (w),

1024 (w), 995 (w), 912 (w), 873 (w), 821 (w), 794 (w), 752 (s), 719 (s), 698 (s), 686 (s), 673

(m), 655 (m). Anal. calcd. for C19H16N4O (316.4): C 72.13, H 5.10, N 17.71. Found: C 72.22,

H 5.20, N 17.61.

Experimental

124

7.6.15. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)butyramide (5dd)

103 mg (0.40 mmol, 40 %) of 5dd was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:10)).

Mp. 126 °C. 1H-NMR (300 MHz, CDCl3): δ = 0.89 (t, 3J = 7.4 Hz, 3 H), 1.57-1.69 (m, 2 H),

2.15 (t, 3J = 7.4 Hz, 2 H), 4.46 (d, 3J = 5.7 Hz, 2 H), 5.48 (s, 2 H), 6.27 (br s, 1 H), 7.24 (m,

1 H), 7.27 (m, 1 H), 7.35-7.40 (m, 3 H), 7.45 (s, 1 H). 13C-NMR (75 MHz, CDCl3): δ = 13.8

(CH3), 19.1 (CH2), 35.0 (CH2), 38.5 (CH2), 54.4 (CH2), 122.2 (CH), 128.2 (CH), 129.0 (CH),

129.3 (CH), 134.6 (Cquat), 145.3 (Cquat), 173.2 (Cquat). EI-MS: m/z (%) = 259 ([M + H]+, 6.8),

258 (M+, 34.5), 240 (3.1), 229 ([M - C2H5]+, 1.7), 215 ([M - C3H7]

+, 1.2), 187 ([M - C4H7O]+,

30.0), 173 (C10H11N3+, 2.2), 167 ([M - C7H7]

+, 2.9), 159 (C9H9N3+, 13.9), 143 (C8H5N3

+, 51.7),

139 (12.4), 130 (4.8), 115 (3.7), 104 (2.5), 97 (16.9), 91 (C7H7+, 100), 83 (3.8), 71 (C4H7O

+,

11.1), 65 (C5H5+, 11.4), 43 (C3H7

+, 19.2). IR (ATR) [cm-1] = 3078 (w), 3035 (w), 2962 (w),

2931 (w), 2873 (w), 2827 (w), 2779 (w), 1631 (s), 1608 (w), 1544 (m), 1494 (w), 1454 (w),

1436 (w), 1404 (w), 1367 (w), 1342 (w), 1334 (w), 1276 (w), 1222 (w), 1209 (w), 1165 (w),

1130 (w), 1109 (w), 1056 (w), 1041 (w), 1028 (w), 1001 (w), 858 (w), 829 (w), 808 (w), 781

(w), 742 (w), 717 (w), 692 (m), 667 (w). Anal. calcd. for C14H18N4O (258.3): C 65.09, H 7.02,

N 21.69. Found C 65.34, H 7.01, N 21.76.

Experimental

125

7.6.16. N-(2-(((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)amino)-2-oxoethyl)-2,2,2-trifluoro

acetamide (5ee)

208 mg (0.61 mmol, 61 %) of 5ee was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:20)).

Mp. 177 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 3.81 (d, 3J = 4.3 Hz , 2 H), 4.32 (d, 3 J = 5.6

Hz, 2 H), 5.57 (s, 2 H), 7.31-7.41 (m, 5H), 7.98 (s, 1H), 8.57 (t, 3J = 5.4 Hz, 1H), 9.63 (br s,

1H). 13C-NMR (75 MHz, d6-DMSO): δ = 34.3 (CH2), 41.9 (CH2), 52.8 (CH2), 115.9 (d, 1J =

287.9 Hz, Cquat), 123.1 (CH), 128.0 (CH), 128.14 (CH), 128.8 (CH), 136.1 (Cquat), 144.8

(Cquat), 156.6 (d, 2J = 36.4 Hz, Cquat), 166.96 (Cquat). EI-MS: m/z (%) = 343 ([M + 2H]+, 3.2),

342 ([M + H]+, 16.3), 341 (M+, 25.8), 299 ([M - 2HF - 2H]+, 8.5), 187 ([M - C4H3F3NO2]+, 11.9),

172 ([M - C4H4F3N2O2]+, 1.9), 159 (C9H9N3

+, 2.5), 144 (C8H6N3+, 12.1), 130 (3.8), 126

(C3H3F3NO+, 9.0), 104 (2.7), 97 (C2F3O+, 9.0), 91 (C7H7

+, 100), 65 (C5H5+, 8.7), 43 (C3H7

+,

2.7), 39 (C3H3+, 2.9). IR (ATR) [cm-1] = 3068 (w), 2947 (w), 2924 (w), 2906 (w), 2854 (w),

1701 (s), 1658 (s), 1558 (m), 1544 (m), 1494 (w), 1456 (w), 1427 (w), 1382 (w), 1359 (w),

1327 (w), 1282 (w), 1242 (w), 1213 (m), 1184 (s), 1161 (s), 1132 (w), 1089 (w), 1076 (w),

1058 (w), 692 (m), 1028 (w), 1004 (w), 844 (w), 763 (w), 742 (w), 723 (m), 713 (m), 659 (w).

Anal. calcd. for C14H14F3N5O2 (341.3): C 49.27, H 4.13, N 20.52. Found C 49.42, H 4.35, N

20.33.

Experimental

126

7.6.17. 3-Phenyl-N-((1-((phenylthio)methyl)-1H-1,2,3-triazol-4-yl)methyl)propanamide

(5ff)

222 mg (0.63 mmol, 63 %) of 5ff was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:10)).

Mp. 75 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 2.36 (t, 3J = 7.8 Hz, 2 H), 2.78 (t, 3J = 7.8 Hz,

2 H), 4.22 (d, 3J = 5.7 Hz, 2 H), 5.88 (s, 2 H), 7.14-7.41 (m, 10 H), 7.22 (s, 1 H), 8.32 (br t, 3J

= 5.60 Hz, 1 H). 13C-NMR (75 MHz, d6-DMSO): δ = 31.0 (CH2), 34.0 (CH2), 36.8 (CH2), 51.4

(CH2), 122.4 (CH), 125.9 (CH), 127.5 (CH), 128.2 (CH), 128.2 (CH), 129.2 (CH), 130.3 (CH),

132.6 (Cquat), 141.2 (Cquat), 145.5 (Cquat), 171.3 (Cquat). EI-MS: m/z (%) = 353 ([M + H]+, 3.3),

352 (M+, 12.6), 234 (13.1), 215 (5.8), 188 (C12H14NO+, 12.2), 149 (C9H11NO+, 9.3), 150

(C9H12NO+, 4.3), 134 (C9H10O+, 2.2), 133 (C9H9O

+, 2.9), 125 (C4H5N4O+, 100), 123 (C7H7S

+,

18.9), 105 (C8H9+, 17.3), 91 (C7H7

+, 30.0), 83 (C4H5NO+, 15.5), 77 (C6H5+, 12.6), 68

(C2H2N3+, 11.1), 54 (C3H4N

+, 41.4). IR (ATR) [cm-1] = 3059 (w), 3003 (w), 2953 (w), 2926

(w), 2852 (w), 1635 (s), 1583 (w), 1543 (s), 1494 (w), 1483 (w), 1454 (w), 1438 (w), 1404

(w), 1386 (w), 1375 (w), 1334 (w), 1309 (w), 1278 (w), 1261 (w), 1226 (m), 1184 (w), 1161

(w), 1111 (w), 1085 (w), 1047 (m), 1026 (m), 993 (w), 968 (w), 827 (w), 777 (w), 740 (s), 721

(m), 692 (s), 659 (w), 613 (w). Anal. calcd. for C19H20N4OS (352.5): C 64.75, H 5.72, N

15.90, S 9.10. Found C 64.62, H 5.71, N 16.19, S 9.11.

Experimental

127

7.6.18. 2-(Phenylamino)-N-((1-((phenylthio)methyl)-1H-1,2,3-triazol-4-yl)methyl)

acetamide (5gg)

276 mg (0.78 mmol, 78 %) of 5gg was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:20)).

Mp. 97 °C. 1H-NMR (300 MHz, d6-acetone): δ = 3.74 (d, 3J = 4.9 Hz, 2 H), 4.43 (m, 2 H),

5.39 (br s, 1 H), 5.81 (s, 2 H), 6.56-6.67 (m, 3 H), 7.07-7.14 (m, 2 H), 7.27-7.43 (m, 5 H),

7.69 (s, 1 H), 7.78 (br s, 1 H). 13C-NMR (75 MHz, d6-acetone): δ = 35.3 (CH2), 48.6 (CH2),

53.5 (CH2), 113.6 (CH), 118.2 (CH), 122.8 (CH), 128.9 (CH), 129.9 (CH), 130.2 (CH), 132.4

(CH), 133.7 (Cquat), 146.7 (Cquat), 149.2 (Cquat), 171.1 (Cquat). EI-MS: m/z (%) = 355

([M + 2H]+, 2.8), 354 ([M + H]+, 8.9), 353 (M+, 36.5), 230 (4.6), 228 (7.8), 221 (C10H13N4S+,

25.3), 205 (C10H11N3S+, 13.0), 167 (2.0), 151 (5.6), 149 (8.1), 133 (6.5), 123 (C7H7S

+, 38.2),

106 (C7H8N+, 100), 83 (5.7), 77 (C6H5

+, 21.3), 51 (6.1), 43 (C2H3O+, 9.0), 39 (C3H3

+, 3.4). IR

(ATR) [cm-1] = 3383 (w), 3302 (w), 3126 (w), 2980 (w), 2970 (w), 2889 (w), 1637 (m), 1604

(m), 1552 (w), 1512 (m), 1485 (w), 1471 (w), 1436 (w), 1421 (w), 1396 (w), 1357 (w), 1338

(w), 1317 (m), 1288 (w), 1257 (w), 1232 (w), 1180 (w), 1155 (w), 1111 (w), 1074 (w), 1045

(m), 1020 (w), 877 (w), 854 (w), 788 (w), 746 (s), 723 (w), 686 (m), 651 (w). Anal. calcd. for

C18H19N5OS (353.4): C 61.17, H 5.42, N 19.81, S 9.07. Found: C 61.06, H 5.41, N 19.51, S

9.17.

Experimental

128

7.6.19. 2-(Phenylthio)-N-((1-((phenylthio)methyl)-1H-1,2,3-triazol-4-yl)methyl)

acetamide (5hh)

218 mg (0.59 mmol, 59 %) of 5hh was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:10)).

Mp. 102 °C. 1H-NMR (300 MHz, d6-acetone): δ = 3.68 (s, 2 H), 4.40 (d, 3J = 5.8 Hz, 2 H),

5.81 (s, 2 H), 7.17-7.42 (m, 10 H), 7.60 (s, 1 H), 7.85 (br s, 1 H). 13C-NMR (75 MHz, d6-

acetone): δ = 35.8 (CH2), 37.8 (CH2), 53.5 (CH2), 122.8 (CH), 127.1 (CH), 128.9 (CH), 129.4

(CH), 129.9 (CH), 130.2 (CH), 132.3 (CH), 133.7 (Cquat), 136.9 (Cquat), 146.4 (Cquat), 168.6

(Cquat). EI-MS: m/z (%) = 372 ([M + 2H]+, 5.0), 371 ([M + H]+, 10.8), 370 (M+, 45.2), 261 (3.8),

247 (8.9), 221 (C10H13N4S+, 71.6), 205 (4.4), 176 (2.9), 151 (2.2), 147 (6.1), 135 (2.0), 125

(C7H9S+, 14.6), 123 (C7H7S

+, 100), 111 (C6H7S+, 13.2), 109 (C6H5S

+, 15.0), 96 (7.6), 83

(C4H5NO+, 24.3), 84 (C4H6NO+, 9.7), 65 (C5H5+, 6.7), 54 (C3H4N

+, 10.0), 45 (26.2), 39 (C3H3+,

6.7). IR (ATR) [cm-1] = 3008 (w), 2924 (w), 2902 (w), 2852 (w), 2681 (w), 1645 (s), 1517

(m), 1481 (w), 1471 (w), 1436 (w), 1408 (w), 1392 (w), 1332 (w), 1284 (w), 1257 (w), 1240

(w), 1230 (w), 1217 (w), 1049 (w), 1026 (w), 759 (m), 732 (s), 752 (w), 690 (s), 665 (w),

630 (w). Anal. calcd. for C18H18N4OS2 (370.5): C 58.35, H 4.90, N 15.12, S 17.31. Found: C

58.50, H 4.95, N 15.07, S 17.18.

Experimental

129

7.6.20. N-((1-((Phenylthio)methyl)-1H-1,2,3-triazol-4-yl)methyl)furan-2-carboxamide

(5ii)

267 mg (0.85 mmol, 85 %) of 5ii was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (1:1), product

obtainment with n-hexane:ethylacetate (1:20)).

Mp. 138 °C. 1H-NMR (300 MHz, d6-acetone): δ = 4.55-4.58 (m, 2 H), 5.84 (s, 2 H), 6.58 (dd,

3J = 1.8 Hz, 1 H), 7.06 (dd, 3J = 2.7 Hz, 4J = 0.8 Hz, 1 H), 7.25-7.34 (m, 3 H), 7.35-7.42 (m,

2 H), 7.66 (dd, 3J = 1.8 Hz, 4J = 0.8 Hz, 1 H), 7.80 (s, 1 H), 8.01 (br s, 1 H). 13C-NMR

(75 MHz, d6-acetone): δ = 35.2 (CH2), 53.5 (CH2), 112.6 (CH), 114.3 (CH), 123.2 (CH),

128.9 (CH), 130.1 (CH), 132.5 (CH) 133.7 (Cquat), 145.4 (CH), 146.6 (Cquat), 149.4 (Cquat),

158.7 (Cquat). EI-MS: m/z (%) = 315 ([M + H]+, 1.8), 314 (M+, 7.3), 268 (2.8), 204 (4.7), 205

(C10H11N3S+, 23.6), 206 (2.7), 177 ([M - N2 - C6H5S]+ , 22.3), 191 (1.3), 148 ([M - C7H8N3S]+,

11.4), 136 (1.8), 123 (C6H5NO2+, 10.7), 124 (C6H6NO2

+, 7.8), 109 (6.1), 95 (C5H3O2+, 100),

84 (5.6), 67 (3.1), 55 (3.6), 45 (5.2), 39 (C3H3+, 9.4). IR (ATR) [cm-1] = 3051 (w), 3010 (w),

2954 (w), 2918 (w), 2837 (w), 2779 (w), 1643 (s), 1595 (m), 1571 (w), 1539 (m), 1469 (w),

1427 (w), 1402 (w), 1355 (w), 1303 (m), 1282 (m), 1236 (m), 1226 (w), 1188 (m), 1122 (m),

1053 (s), 1006 (m), 981 (w), 910 (w), 885 (w), 837 (w), 794 (w), 769 (w), 759 (s), 744 (s),

713 (m), 690 (s). Anal. calcd. for C15H14N4O2S (314.4): C 57.31, H 4.49, N 17.82, S 10.20.

Found: C 57.43, H 4.56, N 17.60, S 10.23.

Experimental

130

7.7. General Procedure for Chemoenzymatic One-pot Amidation-Coupling Sequence

To a solution of 55 mg (1.00 mmol) of propargylamine (2), in 2.0 mL dry MTBE in a screw-

cap Schlenk vessel, were added 1.20 mmol of ester 1 and Novozyme® 435 (50 % w/w of

respective ester substrate 1) and reaction was allowed to shake for 4-24 h (depending upon

the nature of ester 1 used) in an incubating shaker at 45 °C. After the completion of

aminolysis, 2.0 mL of DMF was added in the same reaction vessel and reaction mixture was

flushed with argon for 15 min. Then 1.00 mmol of respective iodoaryl 4h-m, 115 mg (1.00

mmol) of TMG, 23 mg (2 mol%) of Pd(PPh3)4 and 8 mg (4 mol%) of CuI were added to the

reaction mixture under argon and the reaction was allowed to shake for 1 h at 45 °C. After 1

h of reaction time, the reaction mixture was filtered to remove the enzyme beads. Then, to

the filtered reaction mixture, was added 5.0 mL of brine followed by extraction with

ethylacetate (3 x 10.0 mL). The combined organic layers were dried with anhydrous Na2SO4

and pure product was obtained after column chromatography on silica gel using n-

hexane:ethylacetate as eluent to get analytically pure product 6.

Table 7.4. Experimental details of chemoenzymatic one-pot synthesis of coupled product 6.

Entry Ester Substrate 1 Iodoaryl

(4h-m)

Coupled Product 6

1 233 mg (1.20 mmol)

of 1a

204 mg (1.00 mmol)

of 4h

170 mg (58 %)

of 6ca

2 233 mg (1.20 mmol)

of 1a

234 mg (1.00 mmol)

of 4i

200 mg (62 %)

of 6da

3 197 mg (1.20 mmol)

of 1b

204 mg (1.00 mmol)

of 4h

142 mg (54 %)

of 6ea

4 180 mg (1.20 mmol)

of 1c

204 mg (1.00 mmol)

of 4h

132 mg (53 %)

of 6fa

5 180 mg (1.20 mmol)

of 1c

262 mg (1.00 mmol)

of 4j

227 mg (74 %)

of 6ga

6 151 mg (1.20 mmol)

of 1s

210 mg (1.00 mmol)

of 4k

164 mg (71 %)

of 6ha

7 151 mg (1.20 mmol)

of 1s

222 mg (1.00 mmol)

of 4l

151 mg (62 %)

of 6ia

8 171 mg (1.20 mmol)

of 1t

210 mg (1.00 mmol)

of 4k

109 mg (44 %)

of 6ja

aReaction time of 24 h for Novozyme

® 435 catalyzed aminolysis.

bReaction time of 4 h for Novozyme

®

435 catalyzed aminolysis.

Experimental

131

Entry Ester Substrate 1 Iodoaryl

4g-4n

Coupled Product 6

9 205 mg (1.20 mmol)

of 1u

246 mg (1.00 mmol)

of 4m

191 mg (64 %)

of 6kb

10 192 mg (1.20 mmol)

of 1aa

204 mg (1.00 mmol)

of 4h

132 mg (51 %)

of 6la

11 222 mg (1.20 mmol)

of 1dd

204 mg (1.00 mmol)

of 4h

202 mg (71 %)

of 6ma

aReaction time of 24 h for Novozyme

® 435 catalyzed aminolysis.

bReaction time of 4 h for

Novozyme®

435 catalyzed aminolysis.

7.8. Analytical Data of Arylated Propargylamides 6

7.8.1. 3-(4-Methoxyphenyl)-N-(3-phenylprop-2-yn-1-yl)propanamide (6c)

170 mg (0.58 mmol, 58 %) of 6c was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (5:1)).

Mp. 101 °C. 1H-NMR (300 MHz, CDCl3): δ = 2.48 (t, 3J = 7.6 Hz, 2 H), 2.93 (t, 3J= 7.6 Hz,

2 H), 3.75 (s, 3 H), 4.24 (d, 3J = 5.2 Hz, 2 H), 5.63 (br s, 1 H), 6.81 (d, 3J = 8.7 Hz, 2 H), 7.12

(d, 3J = 8.7 Hz, 2 H), 7.28-7.43 (m, 5H). 13C-NMR (75 MHz , CDCl3): δ = 30.1 (CH2), 30.9

(CH2), 38.7 (CH2), 55.3 (CH3), 83.5 (Cquat), 84.9 (Cquat), 114.1 (CH), 122.7 (Cquat), 128.5 (CH),

128.6 (CH), 129.5 (CH), 131.8 (CH), 132.8 (Cquat), 158.2 (Cquat), 171.9 (Cquat). EI-MS: m/z

(%) = 294 ([M+H]+, 2.7), 293 (M+, 13.7), 292 ([M - H]+, 12.4), 216 ([M - C6H5]+, 1.7), 172 ([M -

C8H9O]+, 100), 135 (C9H11O+, 20.8), 121 (C8H9O

+, 61.4), 115 (C9H7+, 18.2), 105 (C7H5O

+,

12.9), 77 (C6H5+, 11.7), 43 (C3H7

+, 4.9). IR (ATR) [cm-1] = 3059 (w), 2995 (w), 2954 (w),

2937 (w), 2922 (w), 2906 (w), 2866 (w), 2835 (w), 1635 (s), 1610 (w), 1535 (m), 1508 (w),

1489 (w), 1446 (w), 1421 (w), 1377 (w), 1340 (w), 1319 (w), 1300 (w), 1251 (m), 1240 (s),

1211 (w), 1180 (w), 1157 (w), 1111 (w), 1078 (w), 1035 (m), 1020 (w), 941 (w), 914 (w), 883

Experimental

132

(w), 831 (m), 813 (w), 759 (s), 713 (w), 690 (s), 661 (m). Anal. calcd. for C19H19NO2 (293.4):

C 77.79, H 6.53, N 4.77. Found C 77.56, H 6.38, N 4.68.

7.8.2. 3-(4-Methoxyphenyl)-N-(3-(4-methoxyphenyl)prop-2-yn-1-yl)propanamide (6d)

200 mg (0.62 mmol, 62 %) of 6d was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (5:1)).

Mp. 114 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 2.37 (t, 3J = 7.7 Hz, 2 H), 2.76 (t, 3J = 7.5

Hz, 2 H), 3.68 (s, 3 H), 3.76 (s, 3 H), 4.06 (d, 3J = 5.4, 2 H), 6.80 (d, 3J = 8.7 Hz, 2 H), 6.92

(d, 3J = 8.8 Hz, 2 H), 7.10 (d, 3J = 8.7 Hz, 2 H), 7.34 (d, 3J = 8.8 Hz, 2 H), 8.32 (t, 3J = 5.3 Hz,

1 H). 13C-NMR (75 MHz, d6-DMSO): 28.5 (CH2), 30.1 (CH2), 37.1 (CH2), 54.9 (CH3), 55.2

(CH3), 81.5 (Cquat), 85.5 (Cquat), 113.7 (CH), 114.3 (CH, Cquat), 129.2 (CH), 132.9 (CH), 133.1

(Cquat), 157.5 (Cquat), 159.3 (Cquat), 171.2 (Cquat). EI-MS: m/z (%) = 324 ([M + H]+, 3.1), 323

(M+, 15.2), 281 (1.3), 221 (1.6), 202 ([M - C8H9O]+, 100), 188 (C11H10NO2+, 4.2), 163

(C10H11O2+, 2.5), 162 (C10H12NO+, 10.1), 161 (C10H9O2

+, 20.2), 145 (C10H9O+, 15.6), 135

(C9H11O+, 23.6), 121 (C8H9O

+, 36.4), 102 (4.8), 91 (C7H7+, 6.4), 89 (2.3), 77 (C6H5

+, 5.9), 73

(2.0), 43 (C3H7+, 5.0), 40 (3.6). IR (ATR) [cm-1] = 3032 (w), 2962 (w), 2920 (w), 2843 (w),

1633 (m), 1604 (w), 1529 (w), 1510 (m), 1481 (w), 1462 (w), 1440 (w), 1417 (w), 1373 (w),

1344 (w), 1294 (w), 1244 (s), 1220 (w), 1176 (w), 1149 (w), 1101 (m), 1076 (m), 1029 (s),

1016 (s), 1006 (m), 939 (w), 923 (w), 860 (w), 846 (w), 802 (s), 790 (s), 767 (m), 759 (m),

729 (w), 692 (m), 651 (w), 640 (w), 623 (w). Anal. calcd. for C20H21NO3 (323.4): C 74.28, H

6.55, N 4.33. Found C 74.41, H 6.47, N 4.36.

Experimental

133

7.8.3. 3-Phenyl-N-(3-phenylprop-2-yn-1-yl)propanamide (6e)

142 mg (0.54 mmol, 54 %) of 6e was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (5:1)).

Mp. 102 °C. 1H-NMR (300 MHz, d6-acetone): δ = 2.51 (t, 3J = 8.1 Hz, 2 H), 2.93 (t, 3J = 8.2

Hz, 2 H), 4.21 (d, 3J = 5.4 Hz, 2 H), 7.13-7.29 (m, 5 H), 7.34-7.43 (br m, 5 H), 7.50 (br s,

1 H). 13C-NMR (75 MHz, d6-acetone): δ = 29.8 (CH2), 32.3 (CH2), 38.3 (CH2), 82.7 (Cquat),

87.3 (Cquat), 124.0 (Cquat), 126.9 (CH), 129.3 (CH), 129.3 (CH), 129.4 (CH), 132.4 (CH),

142.5 (Cquat), 172.0 (Cquat). EI-MS: m/z (%) = 264 ([M + H]+, 3.5), 263 (M+, 17.2), 262 ([M -

H]+, 3.6), 203 (2.5), 172 ([M - C7H7]+, 100), 158 ([M - C8H9]

+, 5.3), 130 (C9H8N+, 30.2), 132

(C9H10N+, 5.5), 115 (C9H7

+, 17.1), 105 (C8H9+, 18.2), 91 (C7H7

+, 19.4), 77 (C6H5+, 7.6), 65

(C5H5+, 3.6). IR (ATR) [cm-1] = 3028 (w), 2927 (w), 2862 (w), 1633 (s), 1533 (s), 1489 (w),

1429 (w), 1379 (w), 1346 (w), 1303 (w), 1294 (w), 1263 (w), 1226 (m), 1072 (w), 1028 (w),

1012 (w), 754 (s), 688 (s), 609 (s). Anal. calcd. for C18H17NO (263.3): C 82.10, H 6.51,

N 5.32. Found C 81.99, H 6.55, N 5.31.

Experimental

134

7.8.4. 2-Phenyl-N-(3-phenylprop-2-yn-1-yl)acetamide (6f)

132 mg (0.53 mmol, 53 %) of 6f was obtained as colorless solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (5:1)).

Mp. 96 °C. 1H-NMR (300 MHz, CDCl3): δ = 3.62 (s, 2 H), 4.24 (d, 3J = 5.3 Hz, 2 H), 5.70 (br

s, 1 H), 7.27-7.41 (br m, 10 H). 13C-NMR (75 MHz , CDCl3): δ = 30.3 (CH2), 43.7 (CH2), 83.5

(Cquat), 84.7 (Cquat), 122.6 (Cquat), 127.6 (CH), 128.4 (CH), 128.6 (CH), 129.2 (CH), 129.6

(CH), 131.8 (CH), 134.6 (Cquat), 170.7 (Cquat). EI-MS: m/z (%) = 250 ([M + H]+, 7.1), 249 (M+,

36.6), 248 ([M - H]+, 2.0), 174 (11.9), 158 ([M - C7H7]+, 100), 130 (C9H8N

+, 49.4), 119

(C8H7O+, 28.7), 115 (C9H7

+, 53.2), 91 (C7H7+, 98.3), 77 (C6H5

+, 14.0), 65 (C5H5+, 16.9), 43

(C3H7+, 8.9). IR (ATR) [cm-1] = 3026 (w), 2939 (w), 2914 (w), 2856 (w), 2773 (w), 2735 (w),

2601 (w), 1946 (w), 1874 (w), 1805 (w), 1637 (s), 1598 (w), 1537 (s), 1490 (w), 1452 (w),

1442 (w), 1413 (w), 1386 (w), 1365 (w), 1330 (w), 1292 (w), 1278 (w), 1242 (w), 1232 (w),

1199 (w), 1182 (w), 1153 (w), 1120 (w), 1097 (w), 1070 (w), 1049 (w), 1014 (w), 999 (w),

968 (w), 939 (w), 904 (w), 871 (w), 829 (w), 802 (w), 771 (w), 754 (s), 721 (m), 686 (s), 623

(w). Anal. calcd. for C17H15NO (249.3): C 81.90, H 6.06, N 5.62. Found C 82.08, H 6.11, N

5.58.

Experimental

135

7.8.5. Methyl 4-(3-(2-phenylacetamido)prop-1-yn-1-yl)benzoate (6g)

227 mg (0.74 mmol, 74 %) of 6g was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (3:1)).

Mp. 147 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 3.47 (s, 2 H), 3.85 (s, 3 H), 4.16 (d, 3J = 5.3

Hz, 2 H), 7.22-7.33 (m, 5 H), 7.54 (d, 3J = 8.3 Hz, 2 H), 7.94 Hz (d, 3J = 8.3 Hz, 2 H), 8.64 (t,

3J = 5.0 Hz, 1 H). 13C-NMR (75 MHz, d6-DMSO): δ = 28.8 (CH2), 42.0 (CH2), 52.3 (CH3),

80.9 (Cquat), 90.4 (Cquat), 126.4 (CH2), 127.0 (Cquat), 128.2 (CH), 129.0 (CH), 129.2 (Cquat),

129.4 (CH), 131.7 (CH), 136.0 (Cquat), 165.6 (Cquat), 170.0 (Cquat). EI-MS: m/z (%) = 307 (M+,

3.4), 293 ([M - CH3 + 1H]+, 13.9), 275 ([M - MeOH]+, 1.6), 263 ([M - CO2]+, 7.7), 216 ([M -

C7H7]+, 1.7), 199 (2.3), 184 (2.7), 183 (8.7), 174 (3.6), 167 (17.3), 150 (C9H12NO+, 10.3), 149

(C9H11NO•+, 100), 127 (13.9), 111 (3.3), 97 (7.5), 91 (C7H7+, 7.6), 85 (C4H5O2

+, 16.9), 83

(C4H3O2+, 7.5), 77 (C6H5

+, 2.5), 71 (C3H3O2+, 28.3), 69 (C3HO2

+, 11.3), 65 (C5H5+, 2.3), 43

(C3H7+, 19.1), 39 (C3H3

+, 1.6). IR (ATR) [cm-1] = 3068 (w), 2954 (w), 2924 (w), 2850 (w),

2821 (w), 1716 (m), 1639 (m), 1598 (w), 1543 (w), 1490 (w), 1454 (w), 1436 (w), 1408 (w),

1328 (w), 1303 (w), 1273 (m), 1257 (m), 1228 (w), 1192 (w), 1174 (w), 1147 (w), 1109 (w),

1099 (w), 1080 (w), 1024 (w), 1014 (w), 980 (w), 916 (w), 908 (w), 860 (w), 848 (w), 829 (w),

765 (w), 746 (w), 732 (w), 694 (s), 642 (w). Anal. calcd. for C19H17NO3 (307.3): C 74.25, H

5.58, N 4.56. Found C 74.03, H 5.39, N 4.48.

Experimental

136

7.8.6. N-(3-(Thiophen-2-yl)prop-2-yn-1-yl)furan-2-carboxamide (6h)

164 mg (0.71 mmol, 71 %) of 6h was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (6:1)).

Mp. 67 °C. 1H-NMR (300 MHz, CDCl3): δ = 4.47 (d, 3J = 5.4 Hz, 2 H), 6.50 (dd, 3J = 3.5 Hz,

4J = 1.8 Hz, 1 H), 6.62 (br s, 1 H), 6.95 (dd, 3J = 5.2 Hz, 3J = 3.7 Hz, 1 H), 7.15 (dd, 3J = 3.5

Hz, 4J = 0.8 Hz, 1 H), 7.20 (m, 1 H), 7.24 (m, 1 H), 7.45 (m, 1 H). 13C-NMR (75 MHz, CDCl3):

δ = 29.9 (CH2), 77.1 (Cquat), 88.6 (Cquat), 112.3 (CH), 114.9 (CH), 122.5 (Cquat), 127.1 (CH),

127.4 (CH), 132.5 (CH), 144.3 (CH), 147.6 (Cquat), 158.0 (Cquat). EI-MS: m/z (%) = 232 ([M +

H]+, 2.8), 231 (M+, 17.7), 230 ([M - H]+, 3.9), 202 ([M - CHO]+, 100), 186 ([M - CH2S]+, 2.7),

184 (10.4), 174 (C10H8NO2+, 8.2), 170 (1.2), 159 (3.3), 148 ([M - C4H3S]+, 2.1), 136

(C7H6NS+, 8.6), 134 (C7H4NS+, 2.9), 121 (C7H5S+, 8.9), 109 (19.0), 95 (C5H3O2

+, 55.6),

77 (7.9), 69 (5.1), 45 (3.6). IR (ATR) [cm-1] = 3076 (w), 3041 (w), 2968 (w), 2935 (w), 2779

(w), 1672 (w), 1651 (m), 1591 (m), 1523 (m), 1477 (w), 1381 (w), 1357 (w), 1348 (w), 1294

(w), 1257 (w), 1226 (w), 1186 (s), 1143 (w), 1082 (w), 1307 (w), 1008 (m), 987 (w), 935 (w),

906 (w), 885 (w), 848 (w), 831 (w), 825 (w), 781 (w), 750 (m), 692 (s), 607 (m). Anal. calcd.

for C12H9NO2S (231.3): C 62.32, H 3.92, N 6.06, S 13.86. Found C 62.40, H 3.95, N 6.05, S

14.11.

Experimental

137

7.8.7. N-(3-(5-Formylfuran-2-yl)prop-2-yn-1-yl)furan-2-carboxamide (6i)

151 mg (0.62 mmol, 62 %) of 6i was obtained as yellow solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (3:1)).

Mp. 118 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 4.34 (d, 3J = 5.7, 2 H), 6.64 (dd, 3J = 3.5 Hz,

4J = 1.7 Hz, 1 H), 7.03 (d, 3J = 3.7 Hz, 1 H), 7.16 (dd, 3J = 3.5 Hz, 4J = 0.7 Hz, 1 H), 7.56 (d,

3J = 3.7 Hz, 1 H), 7.87 (m, 1 H), 8.99 (t, 3J = 5.6 Hz, 1 H), 9.56 (s, 1 H). 13C-NMR (75 MHz,

d6-DMSO): δ = 28.6 (CH2), 71.0 (Cquat), 95.0 (Cquat), 112.0 (CH), 114.1 (CH), 117.7 (CH),

123.4 (CH), 140.1 (Cquat), 145.5 (CH), 147.3 (Cquat), 152.1 (Cquat), 157.6 (Cquat), 178.1 (Cquat).

EI-MS: m/z (%) = 244 ([M + H]+, 5.1) , 243 (M+, 37.3), 214 ([M - CHO]+, 64.0), 215 ([M - CO]+,

10.0), 186 ([M - C2HO2]+, 39.0), 168 (11.9), 148 (C8H6NO2

+, 12.9), 140 (8.3), 131 (5.6), 130

(11.5), 121 (C7H5O2+, 5.3), 115 (10.1), 103 (11.9), 95 (C5H3O2

+, 100), 77 (5.6), 75 (7.0), 67

(C4H3O+, 6.1), 63 (5.1), 43 (C3H7

+, 5.4), 39 (C3H3+, 13.1). IR (ATR) [cm-1] = 3103 (w), 2985

(w), 2825 (w), 1668 (m), 1647 (m), 1597 (w), 1521 (w), 1506 (m), 1471 (m), 1408 (w), 1390

(m), 1348 (w), 1309 (w), 1284 (w), 1267 (w), 1230 (w), 1188 (w), 1147 (w), 1103 (w), 1080

(w), 1053 (w), 1033 (m), 1020 (m), 1006 (w), 985 (w), 966 (m), 916 (w), 896 (w), 812 (s), 798

(w), 775 (w), 756 (s), 659 (w), 626 (w), 601 (w). Anal. calcd. for C13H9NO4 (243.2): C 64.20,

H 3.73, N 5.76. Found C 64.12, H 4.02, N 5.82.

Experimental

138

7.8.8. N-(3-(Thiophen-2-yl)prop-2-yn-1-yl)thiophene-2-carboxamide (6j)

109 mg (0.44 mmol, 44 %) of 6j was obtained as colorless solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (6:1)).

Mp. 120 °C. 1H-NMR (300 MHz, CDCl3): δ = 4.48 (d, 3J = 5.3 Hz, 2 H), 6.36 (br, 1 H), 6.96

(dd, 3J = 5.2 Hz, 3.7 Hz, 1 H), 7.08 (dd, 3J = 5.0 Hz, 3.8 Hz, 1 H), 7.20 (dd, 3J = 3.6 Hz, 4J =

1.9 Hz, 1 H), 7.24 (dd, 3J = 5.2 Hz, 4J = 1.1 Hz, 1 H), 7.49 (dd, 3J = 5.0 Hz, 4J = 1.1 Hz, 1 H),

7.56 (dd, 3J = 3.7 Hz, 4J = 1.1 Hz, 1 H). 13C-NMR (75 MHz, CDCl3): δ = 30.8 (CH2), 77.4

(Cquat), 88.7 (Cquat), 122.5 (Cquat), 127.1 (CH), 127.5 (CH), 127.8 (CH), 128.7 (CH), 130.5

(CH), 132.6 (CH), 138.3 (Cquat), 161.6 (Cquat). EI-MS: m/z (%) = 247 (M+, 8.5), 246 ([M - H]+,

4.6), 202 ([M - CH2S + H]+, 45.7), 186 (6.0), 183 (4.9), 136 (C7H6NS+, 9.8), 121 (C7H5S+,

10.6), 111 (C5H3OS+, 100), 109 (19.8), 108 (8.9), 83 (C4H3S+, 17.8), 69 (11.0), 45 (11.6). IR

(ATR) [cm-1] = 3097 (w), 3010 (w), 2981 (w), 2924 (w), 2872 (w), 2848 (w), 2358 (w), 2127

(w), 1797 (w), 1708 (w), 1624 (m), 1595 (w), 1541 (m), 1512 (w), 1479 (w), 1458 (w), 1417

(w), 1381 (w), 1359 (w), 1344 (w), 1300 (m), 1284 (m), 1253 (m), 1247 (m), 1188 (w), 1143

(m), 1095 (m), 1080 (w), 1060 (w), 1047 (w), 1031 (w), 1020 (w), 968 (w), 954 (w), 906 (w),

885 (w), 854 (m), 842 (m), 827 (w), 785 (w), 771 (w), 752 (w), 719 (m), 700 (s), 657 (w), 634

(m). Anal. calcd. for C12H9NOS2 (247.3). C 58.27, H 3.67, N 5.66, S 25.93. Found C 58.52, H

3.94, N 5.70, S 26.07.

Experimental

139

7.8.9. N-(3-(4-Acetylphenyl)prop-2-yn-1-yl)-2-(piperidin-1-yl)acetamide (6k)

191 mg (0.64 mmol, 64 %) of 6k was obtained as viscous yellow liquid after purification by

silica gel column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (2:1)).

Viscous yellow liquid. 1H-NMR (300 MHz, d6-DMSO): δ = 1.38 (m, 2 H), 1.53 (m, 4 H), 2.37

(t, 3J = 5.0 Hz, 4 H), 2.57 (s, 3 H), 2.91 (s, 2 H), 4.17 (d, 3J = 5.9 Hz, 2 H), 7.53 (d, 3J = 8.5

Hz, 2 H), 7.94 (d, 3J = 8.6 Hz, 2 H), 8.19 (t, 3J = 5.7 Hz, 1 H). 13C-NMR (75 MHz , d6-DMSO):

δ = 23.5 (CH2), 25.4 (CH2), 26.7 (CH3), 28.5 (CH2), 54.1 (CH2), 61.9 (CH2), 80.5 (Cquat), 90.8

(Cquat), 126.9 (Cquat), 128.4 (CH2), 131.5 (CH2), 136.1 (Cquat), 169.6 (Cquat), 197.2 (Cquat). EI-

MS: m/z (%) = 277 (2.9), 155 ([M - C10H7O]+, 2.5), 155 (2.5), 149 (3.4), 113 (2.6), 112 (5.0),

98 (C6H12N+, 100), 84 (C5H10N

+, 8.5), 73 (C3H7NO•+, 20.1), 70 (6.4), 67 (2.6), 43 (C2H3O+,

6.9). IR (ATR) [cm-1] = 2933 (w), 2854 (w), 2810 (w), 2794 (w), 2756 (w), 1678 (s), 1600

(m), 1552 (w), 1504 (m), 1467 (w), 1452 (w), 1402 (w), 1386 (w), 1355 (w), 1334 (w), 1301

(w), 1259 (s), 1178 (w), 1161 (w), 1126 (w), 1111 (w), 1087 (w), 1074 (w), 1039 (w), 1014

(w), 997 (w), 983 (w) 958 (w), 902 (w), 864 (w), 839 (m), 810 (w), 794 (w), 765 (w), 746 (w),

723 (w), 696 (w), 634 (w). Anal. calcd. for C18H22N2O2 (298.4): C 72.46, H 7.43, N 9.39.

Found C 72.15, H 7.31, N 9.02.

Experimental

140

7.8.10. 3-Phenyl-N-(3-phenylprop-2-yn-1-yl)propiolamide (6l)

132 mg (0.51 mmol, 51 %) of 6l was obtained as colorless solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (6:1)).

Mp. 119 °C. 1H-NMR (300 MHz, CDCl3): δ = 4.38 (d, 3J = 5.3 Hz, 2 H), 6.23 (br, 1H), 7.27-

7.62 (br m, 10 H).13C-NMR (75 MHz , CDCl3): δ = 30.6 (CH2), 82.6 (Cquat), 83.8 (Cquat), 84.1

(Cquat), 85.7 (Cquat), 120.1 (Cquat), 122.4 (Cquat), 128.5 (CH), 128.7 (CH), 128.8 (CH), 130.4

(CH), 131.9 (CH), 132.7 (CH), 153.1 (Cquat). EI-MS: m/z (%) = 260 ([M + H]+, 5.2), 259 (M+,

29.4), 258 ([M - H]+, 30.5), 241 (25.7), 230 (74.2), 215 (54.8), 182 ([M - C6H5]+, 15.6), 154

(11.0), 129 (C9H5O+, 100), 130 (C9H8N

+, 11.9), 115 (C9H7+, 12.8), 101 (C8H5

+, 16.1), 89 (6.1),

77 (C6H5+, 11.7), 63 (4.5). IR (ATR) [cm-1] = 3012 (w), 2989 (w), 2929 (w), 2900 (w), 2615

(w), 2594 (w), 2517 (w), 2436 (w), 2222 (w), 2185 (w), 2146 (w), 2100 (w), 2042 (w), 2015

(w), 1984 (w), 1969 (w), 1950 (w), 1897 (w), 1818 (w), 1737 (w), 1718 (w), 1647 (w), 1618

(m), 1597 (w), 1533 (m), 1487 (m), 1442 (w), 1417 (w), 1394 (w), 1384 (w), 1355 (w), 1303

(m), 1273 (w), 1257 (w), 1219 (m), 1178 (w), 1157 (w), 1097 (w), 1070 (w), 1058 (w), 1016

(w), 997 (w), 987 (w), 960 (w), 916 (w), 902 (w), 877 (w), 842 (w), 788 (w), 754 (s), 731 (w),

686 (w), 623 (w), 605 (w). Anal. calcd. for C18H13NO (259.3): C 83.37, H 5.05, N 5.40, Found

C 83.60, H 5.11, N 5.44.

Experimental

141

7.8.11. 2,2,2-Trifluoro-N-(2-oxo-2-((3-phenylprop-2-yn-1-yl)amino)ethyl)

acetamide (6m)

202 mg (0.71 mmol, 71 %) of 6m was obtained as colorless solid after purification by silica

gel column chromatography (elution started with n-hexane:ethylacetate (10:1), product

obtainment with n-hexane:ethylacetate (4:1)).

Mp. 137 °C. 1H-NMR (300 MHz , CDCl3): δ = 4.07 (d, 3J = 4.9 Hz, 2 H), 4.31 (d, 3J = 5.2 Hz,

2 H), 6.45 (br, 1 H), 7.28-7.42 (br m, 5 H), 7.53 (br, 1 H). 13C-NMR (75 MHz , CDCl3): δ =

30.5 (CH2), 42.8 (CH2), 83.7 (Cquat), 84.1 (Cquat), 115.8 (d, 1J = 287.2 Hz, Cquat), 122.3 (Cquat),

128.5 (CH), 128.9 (CH), 131.9 (CH), 157.7 (d, 2J = 37.9 Hz, Cquat), 166.5 (Cquat). EI-MS:

m/z (%) = 209 (3.0), 171 (C4H6F3N2O2+, 100), 172 ([M - C2HF3NO]+, 14.2), 158 (C10H8NO+,

9.1), 143 (15.1), 130 (C9H8N+, 25.1), 115 (C9H7

+, 48.9), 103 (C8H7+, 12.4), 97 (C2F3O

+, 3.1),

89 (6.8), 77 (C6H5+, 7.8), 69 (CF3

+, 7.4), 43 (C3H7+, 1.9). IR (ATR) [cm-1] = 3099 (w), 2916

(w), 2854 (w), 1701 (s), 1654 (s), 1556 (m), 1490 (w), 1442 (w), 1423 (w), 1386 (w), 1355

(w), 1344 (w), 1284 (w), 1213 (m), 1188 (s), 1157 (s), 1103 (w), 1091 (w), 1070 (w), 1041

(w), 1006 (w), 964 (w), 916 (w), 885 (w), 846 (w), 754 (s), 731 (w), 688 (s), 621 (w), 601 (w).

Anal. calcd. for C13H11F3N2O2 (284.2): C 54.93, H 3.90, N 9.86. Found C 54.68, N 3.93, H

9.79.

Experimental

142

7.9. General Procedure for Chemoenzymatic One-pot Triazole ligation to Arylated

Propargylamides (Synthesis of 8a and 8b)

To a solution of 55 mg (1.00 mmol) of propargylamine (2), in 2.0 mL dry MTBE in a screw-

cap Schlenk vessel, were added 1.20 mmol of ester 1 and Novozyme® 435 (50 % w/w of

respective ester substrate 1) and the reaction was allowed to shake for 4-24 h (depending

upon the nature of ester 1 used) in an incubating shaker at 45 °C. After the aminolysis, 2.0

mL of DMF was added in the same reaction vessel and reaction mixture was flushed with

argon for 15 minutes followed by the addition of 300 mg (1.00 mmol) of (4-iodophenyl)-

ethynyl) trimethylsilane (4p), 115 mg (1.00 mmol) of TMG, 23 mg (2 mol %) of Pd(PPh3)4 and

8 mg (4 mol %) of CuI under argon and the reaction was allowed to shake for 1 h at 45 °C.

After 1 h of reaction time, 58 mg (1.00 mmol) of potassium fluoride (KF) was added into the

reaction vial and was allowed to run for 10 minutes after which 133 mg (1.00 mmol) of

benzyl azide (4a) was added. After 1 h of shaking, the reaction mixture was filtered to

remove the enzyme beads. Then, to the filtered reaction mixture, was added 5.0 mL of brine

followed by extraction with ethylacetate (3 x 10.0 mL). The combined organic layers were

dried with anhydrous Na2SO4 followed by column chromatography (n-hexane:ethylacetate) to

get analytically pure product 8.

Table 7.5. Experimental details of chemoenzymatic one-pot synthesis of triazole ligated

product 8.

Entry Ester

Substrate 1

Aryl

Iodide (4p)

Benzyl azide

(4a)

Product 8

1 166 mg

(1.20 mmol) of 1e

300 mg

(1.00 mmol)

133 mg

(1.00 mmol)

245 mg

(58 %) of 8aa

2 192 mg

(1.20 mmol) of 1aa

300 mg

(1.00 mmol)

133 mg

(1.00 mmol)

259 mg

(62 %) of 8bb

aReaction time of 24 h for Novozyme

® 435 catalyzed aminolysis.

bReaction time of 4 h for

Novozyme®

435 catalyzed aminolysis.

Experimental

143

7.10. Analytical Data of Triazole ligated Systems 8

7.10.1. N-(3-(4-(1-Benzyl-1H-1,2,3-triazol-4-yl)phenyl)prop-2-yn-1-yl)-2-phenoxy

acetamide (8a)

245 mg (0.58 mmol, 58 %) of 8a was obtained as yellow solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (4:1), product

obtainment with n-hexane:ethylacetate (1:1)).

Mp. 147 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 4.21 (d, 3J = 5.6 Hz, 2 H), 4.54 (s, 2 H), 5.65

(s, 2 H), 6.97-7.00 (m, 3 H), 7.28-7.39 (br m, 7 H), 7.45 (d, 3J = 8.4 Hz, 2 H), 7.59-7.62 (m,

1 H), 7.86 (d, 3J = 8.4 Hz, 2 H), 8.69 (s, 1 H).13C-NMR (75 MHz, d6-DMSO): δ = 28.5 (CH2),

53.1 (CH2), 66.8 (CH2), 81.3 (Cquat), 87.7 (Cquat), 114.7 (CH), 121.2 (CH), 121.5 (Cquat), 122.1

(CH), 125.3 (CH), 127.9 (CH), 128.2 (CH), 129.5 (CH), 130.7 (Cquat), 132.0 (CH), 135.9

(Cquat), 145.9 (Cquat), 157.6 (Cquat), 167.7 (Cquat). MALDI-MS: m/z = 423 ([M + H]+). IR (ATR)

[cm-1] = 3034 (w), 2922 (w), 2910 (w), 2856 (w), 1662 (s), 1598 (w), 1587 (w), 1519 (m),

1489 (s), 1456 (w), 1435 (w), 1409 (w), 1350 (w), 1286 (w), 1242 (s), 1226 (s), 1170 (w),

1080 (w), 1060 (w), 1047 (w), 1028 (w), 1018 (w), 1001 (w), 835 (m), 798 (m), 756 (s), 721

(s), 657 (w), 603 (w). Anal. calcd. for C26H22N4O2 (422.5): C 73.92, H 5.25, N 13.26. Found

C 74.06, N 5.32, H 13.47.

Experimental

144

7.10.2. N-(3-(4-(1-Benzyl-1H-1,2,3-triazol-4-yl)phenyl)prop-2-yn-1-yl)-3-phenyl

propiolamide (8b)

258 mg (0.62 mmol, 62 %) of 8b was obtained as yellow solid after purification by silica gel

column chromatography (elution started with n-hexane:ethylacetate (4:1), product

obtainment with n-hexane:ethylacetate (1:1)).

Mp. 150 °C. 1H-NMR (300 MHz, d6-DMSO): δ = 4.23 (d, 3J = 5.5 Hz, 2 H), 5.65 (s, 2 H),

7.33-7.61 (br m, 12 H), 7.86 (d, 3J = 8.3 Hz, 2 H), 8.70 (s, 1 H), 9.36 (t, 3J = 5.3 Hz, 1 H).13C-

NMR (75 MHz, d6-DMSO): δ = 30.7 (CH2), 53.1 (CH2), 81.8 (Cquat), 83.4 (Cquat), 83.9 (Cquat),

86.8 (Cquat), 119.6 (Cquat), 122.1 (CH), 123.3 (Cquat), 125.3 (CH), 127.9 (CH), 128.2 (CH),

128.8 (CH), 129.0 (CH), 130.4 (CH), 130.8 (Cquat), 132.0 (CH), 132.2 (CH), 135.9 (Cquat),

145.9 (Cquat), 152.1(Cquat). MALDI-MS: m/z = 417 ([M + H]+). IR (ATR) [cm-1] = 3037 (w),

2993 (w), 2954 (w), 2922 (w), 2360 (w), 2343 (w), 2331 (w), 2225 (w), 1637 (s), 1535 (m),

1419 (w), 1296 (w), 1217 (w), 1190 (w), 1074 (w), 1049 (w), 1020 (w), 977 (w), 844 (w), 800

(m), 754 (s), 713 (m), 686 (m), 617 (w). Anal. calcd. for C27H20N4O (422.5): C 77.87, H 4.84

N 13.45. Found C 77.59 H 4.66 N 13.35.

Molecule Content

145

Molecule Content

1a

1b

1c

1d

1e

1f

1g

1h

1i

1j

1k

1l

1m

1n

1o

1p

1q

1r

Molecule Content

146

1s

1t

1u

1v

1w

1x

1y

1z

1aa

1bb

1cc

1dd

1ee

2

3a

3b

3c

3e

3f

3h

3i

3j

3k

3n

Molecule Content

147

3o, 3p

3q

3r

3s

3t

3u

3aa

4a

4b

4c

4d

4e

4f

4g

4h

4i

4j

4k

4l

4m

4n

4o

4p

5a

Molecule Content

148

5b

5e

5f

5h

5i

5j

5k

5n

5o, 5p

5s

5t

5aa

5dd

5ee

Molecule Content

149

5ff

5gg

5hh

5ii

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

Molecule Content

150

6m

8a

8b

References

151

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